Method for protecting cotton plants from boll weevil infestation

ABSTRACT

A novel gene encoding a Coleopteran inhibitory  Bacillus thuringiensis  insecticidal crystal protein is disclosed. The protein, tIC851, is insecticidally active and provides plant protection from at least cotton boll weevil,  Anthomomus grandis , when applied to plants in an insecticidally effective composition.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecular biology. Methods and compositions comprising DNA sequences, and polypeptides derived from Bacillus thuringiensis for use in insecticidal formulations and the development of transgenic insect-resistant plants are provided. Novel nucleic acids obtained from Bacillus thuringiensis that encode coleopteran-toxic polypeptides are disclosed. Various methods for making and using these nucleic acids, synthetically modified DNA sequences encoding tIC851 polypeptides, and native and synthetic polypeptide compositions are also disclosed. The use of DNA sequences as diagnostic probes and templates for protein synthesis, and the use of polypeptides, fusion proteins, antibodies, and peptide fragments in various insecticidal, immunological, and diagnostic applications are also disclosed, as are methods of making and using nucleic acid sequences in the development of transgenic plant cells comprising the polynucleotides.

1.2 Description of the Related Art

Environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances, in particular when crops of commercial interest are at issue. The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. B. thuringiensis is well known in the art, and is characterized morphologically as a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are aggregations of proteins specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.

There are several toxin categories established based on primary structure information and the degree of toxin similarities to another. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toxins, a generalized mode of action for B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).

Many of the δ-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Schnepf et al. (Microbiol. Mol. Biol. Rev. (1998) 62:775-806) discusses the genes and proteins that were identified in B. thuringiensis prior to 1998, and sets forth the most recent nomenclature and classification scheme as applied to B. thuringiensis insecticidal genes and proteins. Using older nomenclature classification schemes, cry1 genes were deemed to encode lepidopteran-toxic Cry1 proteins, cry2 genes were deemed to encode Cry2 proteins toxic to both lepidopterans and dipterans, cry3 genes were deemed to encode coleopteran-toxic Cry3 proteins, and cry4 genes were deemed to encode dipteran-toxic Cry4 proteins. However, new nomenclature systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. The classification scheme for many known toxins, not including allelic variations in individual proteins, including dendograms and full Bacillus thuringiensis toxin lists is summarized and regularly updated at http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Most of the nearly 200 Bt crystal protein toxins presently known have some degree of lepidopteran activity associated with them. The large majority of Bacillus thuringiensis insecticidal proteins which have been identified do not have coleopteran controlling activity. Therefore, it is particularly important at least for commercial purposes to identify additional coleopteran specific insecticidal proteins.

Cry3 proteins generally display coleopteran activity, however, these generally have limited host range specificity and are not significantly toxic to target pests unless ingested in very high doses. The cloning and expression of the cry3Bb gene has been described (Donovan et al., 1992). This gene codes for a protein of 74 kDa with activity against Coleopteran insects, particularly the Colorado potato beetle (CPB) and the southern corn root worm (SCRW). Improved Cry3Bb proteins have been engineered which display increased toxicity at the same or lower doses than the wild type protein (U.S. Pat. No. 6,023,013; Feb. 8, 2000).

A B. thuringiensis strain, PS201T6, was reported to have activity against WCRW (Diabrotica virgifera virgifera) (U.S. Pat. No. 5,436,002). This strain also had activity against Musca domestica, Aedes aegypti, and Liriomyza trifoli. The vip1A gene, which produces a vegetative, soluble, insecticidal protein, has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 96/10083, 1996). This gene produces a protein of approximately 80 kDa with activity against both WCRW and Northern Corn Root Worm (NCRW). Another toxin protein with activity against coleopteran insects, including WCRW, is Cry1Ia, an 81-kDa polypeptide, the gene encoding which has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 90/13651, 1990).

2.0 SUMMARY OF THE INVENTION

The polypeptide of the present invention and the novel DNA sequences that encode the protein represent a new B. thuringiensis crystal protein and gene, and share only insubstantial sequence homology with any previously identified coleopteran inhibitory endotoxins described in the prior art. Similarly, the B. thuringiensis strains of the present invention comprise novel gene sequences that express a polypeptide having insecticidal activity against coleopteran insects, the cotton boll weevil (Anthonomus grandis Boheman) in particular.

Disclosed and claimed herein is an isolated Bacillus thuringiensis δ-endotoxin polypeptide comprising SEQ ID NO:8. The inventors have identified an insecticidally-active polypeptide comprising the 632 amino acid long sequence of SEQ ID NO:8 which displays insecticidal activity against coleopteran insects. For example, the inventors have shown that a δ-endotoxin polypeptide comprising the sequence of SEQ ID NO:8 has insecticidal activity against boll weevil larvae (BWV), but not against western corn rootworm larvae.

The polypeptide of SEQ ID NO:8 is encoded by a nucleic acid segment comprising at least the open reading frame as shown in SEQ ID NO:7 from nucleotide position 28 through nucleotide position 1923. The invention also discloses compositions and insecticidal formulations that comprise such a polypeptide. Such composition may be a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet of a bacteria cell that comprises a polynucleotide that encodes such a polypeptide. Exemplary bacterial cells that produce such a polypeptide include Bacillus thuringiensis EG4135 and EG4268, deposited with NRRL respectively on Apr. 28, 2000. The composition as described in detail below may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be preparable by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide. Preferably such compositions are obtainable from cultures of Bacillus thuringiensis EG4135 and EG4268 cells. In all such compositions that contain at least one such insecticidal polypeptide, the polypeptide may be present in a concentration of from about 0.001% to about 99% by weight.

An exemplary insecticidal polypeptide formulation may be prepared by a process comprising the steps of culturing Bacillus thuringiensis EG4135 and EG4268 cells under conditions effective to produce the insecticidal polypeptide; and obtaining the insecticidal polypeptide so produced.

For example, the invention discloses and claims a method of preparing a δ-endotoxin polypeptide having insecticidal activity against a coleopteran insect. The method generally involves isolating from a culture of Bacillus thuringiensis EG4135 and EG4268 cells that have been grown under appropriate conditions, the δ-endotoxin polypeptide produced by the cells. Such polypeptides may be isolated from the cell culture or supernatant or from spore suspensions derived from the cell culture and used in the native form, or may be otherwise purified or concentrated as appropriate for the particular application.

A method of controlling a coleopteran insect population is also provided by the invention. The method generally involves contacting the population with an insecticidally-effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO:8. Such methods may be used to kill or reduce the numbers of coleopteran insects in a given area, or may be prophylactically applied to an environmental area to prevent infestation by a susceptible insect. Preferably the insect ingests, or is contacted with, an insecticidally-effective amount of the polypeptide.

Additionally, the invention provides a purified antibody that specifically binds to the insecticidal polypeptide. Also provided are methods of preparing such an antibody, and methods for using the antibody to isolate, identify, characterize, and/or purify polypeptides to which such an antibody specifically binds. Immunological kits and immunodetection methods useful in the identification of such polypeptides and peptide fragments and/or epitopes thereof are provided in detail herein, and also represent important aspects of the present invention.

Such antibodies may be used to detect the presence of such polypeptides in a sample, or may be used as described hereinbelow in a variety of immunological methods. An exemplary method for detecting a δ-endotoxin polypeptide in a biological sample generally involves obtaining a biological sample suspected of containing a δ-endotoxin polypeptide; contacting the sample with an antibody that specifically binds to the polypeptide, under conditions effective to allow the formation of complexes; and detecting the complexes so formed.

For such methods, the invention also provides an immunodetection kit. Such a kit generally contains, in suitable container means, an antibody that binds to the δ-endotoxin polypeptide, and at least a first immunodetection reagent. Optionally, the kit may provide additional reagents or instructions for using the antibody in the detection of δ-endotoxin polypeptides in a sample.

Preparation of such antibodies may be achieved using the disclosed polypeptide as an antigen in an animal as described below. Antigenic epitopes, shorter peptides, peptide fusions, carrier-linked peptide fragments, and the like may also be generated from a whole or a portion of the polypeptide sequence disclosed in SEQ ID NO:8. Particularly preferred peptides are those that comprise at least 10 contiguous amino acids from the sequence disclosed in SEQ ID NO:8.

In another embodiment, the present invention also provides nucleic acid segments that comprise a selected nucleotide sequence region that comprises the polynucleotide sequence of SEQ ID NO:7. In preferred embodiments, this selected nucleotide sequence region comprises a gene that encodes a polypeptide comprising at least SEQ ID NO:8.

Another aspect of the invention relates to a biologically-pure culture of a wild-type B. thuringiensis bacterium selected from the strains EG4135 and EG4268, deposited on Apr. 28, 2000 with the Agricultural Research Culture Collection, Northern Regional Research Laboratory (NRRL), Peoria, Ill. Also deposited was strain sIC8501 which is an E. coli DH5a containing plasmid pIC17501 which contains at least the native B. thuringiensis strain EG4135 tIC851 coding sequence. These strains were deposited under the terms of the Budapest Treaty, and viability statements pursuant to International Receipt Form BP/4 were obtained. B. thuringiensis strains EG4135 and EG4268 are naturally-occurring strains that contain at least one sequence region encoding the 632 amino acid long polypeptide sequence in SEQ ID NO:8.

A further embodiment of the invention relates to a vector comprising a sequence region that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, a recombinant host cell transformed with such a recombinant vector, and biologically-pure cultures of recombinant bacteria transformed with a polynucleotide sequence that encodes the polypeptide disclosed in SEQ ID NO:8. Exemplary vectors, recombinant host cells, transgenic cell lines, and transgenic plants comprising at least a first sequence region that encodes a polypeptide comprising the sequence of SEQ ID NO:8 are described in detail herein.

The present invention also provides transformed host cells, embryonic plant tissue, plant calli, plantlets, and transgenic plants that comprise a selected sequence region that encodes the insecticidal polypeptide. Such cells are preferably prokaryotic or eukaryotic cells such as bacterial, fungal, or plant cells, with exemplary bacterial cells including Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Escherichia, Salmonella, Agrobacterium or Pseudomonas cells.

The plants and plant host cells are preferably monocotyledonous or dicotyledonous plant cells such as corn, wheat, soybean, oat, cotton, rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley, turf grass, pasture grass, berry, fruit, legume, vegetable, ornamental plant, shrub, cactus, succulent, and tree cell.

Transgenic plants of the present invention preferably have incorporated into their genome or transformed into their chloroplast or plastid genomes a selected polynucleotide (or “transgene”), that comprises at least a first sequence region that encodes the insecticidal polypeptide of SEQ ID NO:8. Transgenic plants are also meant to comprise progeny (descendant, offspring, etc.) of any generation of such a transgenic plant. A seed of any generation of all such transgenic insect-resistant plants wherein said seed comprises a DNA sequence encoding the polypeptide of the present invention is also an important aspect of the invention.

Insect resistant, crossed fertile transgenic plants comprising a transgene that encodes the polypeptide of SEQ ID NO:8 may be prepared by a method that generally involves obtaining a fertile transgenic plant that contains a chromosomally incorporated transgene encoding the insecticidal polypeptide of SEQ ID NO:8; operably linked to a promoter active in the plant; crossing the fertile transgenic plant with a second plant lacking the transgene to obtain a third plant comprising the transgene; and backcrossing the third plant to obtain a backcrossed fertile plant. In such cases, the transgene may be inherited through a male parent or through a female parent. The second plant may be an inbred, and the third plant may be a hybrid.

Likewise, an insect resistant hybrid, transgenic plant may be prepared by a method that generally involves crossing a first and a second inbred plant, wherein one or both of the first and second inbred plants comprises a chromosomally incorporated transgene that encodes the polypeptide of SEQ ID NO:8 operably linked to a plant expressible promoter that expresses the transgene. In illustrative embodiments, the first and second inbred plants may be monocot plants selected from the group consisting of: corn, wheat, rice, barley, oats, rye, sorghum, turfgrass and sugarcane.

In related embodiment, the invention also provides a method of preparing an insect resistant plant. The method generally involves contacting a recipient plant cell with a DNA composition comprising at least a first transgene that encodes the polypeptide of SEQ ID NO:8 under conditions permitting the uptake of the DNA composition; selecting a recipient cell comprising a chromosomally incorporated transgene that encodes the polypeptide; regenerating a plant from the selected cell; and identifying a fertile transgenic plant that has enhanced insect resistance relative to the corresponding non-transformed plant.

A method of producing transgenic seed generally involves obtaining a fertile transgenic plant comprising a chromosomally integrated transgene that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, operably linked to a promoter that expresses the transgene in a plant; and growing the plant under appropriate conditions to produce the transgenic seed.

A method of producing progeny of any generation of an insect resistance-enhanced fertile transgenic plant is also provided by the invention. The method generally involves collecting transgenic seed from a transgenic plant comprising a chromosomally integrated transgene that encodes the polypeptide of SEQ ID NO:8, operably linked to a promoter that expresses the transgene in the plant; planting the collected transgenic seed; and growing the progeny transgenic plants from the seed.

These methods for creating transgenic plants, progeny and seed may involve contacting the plant cell with the DNA composition using one of the processes well-known for plant cell transformation such as microprojectile bombardment, electroporation or Agrobacterium-mediated transformation.

An exemplary method disclosed herein provides for protecting a plant from cotton boll weevil infestation comprising providing to a boll weevil in its diet a plant transformed to express a protein toxic to said weevil wherein said protein is expressed in sufficient amounts to control boll weevil infestation and wherein said protein is selected from the group consisting of Cry22Aa, ET70, and tIC851. In a further embodiment of this method, a plant expressing two or more of these proteins for the purpose of reducing boll weevil infestation is contemplated, in particular for reducing the development of races of boll weevils resistant to any of these proteins.

These and other embodiments of the present invention will be apparent to those of skill in the art from the following examples and claims, having benefit of the teachings of the Specification herein.

2.1 tIC851 POLYNUCLEOTIDE SEQUENCES

The present invention provides polynucleotide sequences that can be isolated from Bacillus thuringiensis strains, that are free from total genomic DNA, and that encode the novel insecticidal polypeptides and peptide fragments disclosed herein. The polynucleotides encoding these peptides and polypeptides may encode active insecticidal proteins, or peptide fragments, polypeptide subunits, functional domains, or the like of one or more tIC851 or tIC851-related crystal proteins, such as the polypeptide disclosed in SEQ ID NO:8. In addition the invention encompasses nucleic acid sequences which may be synthesized entirely in vitro using methods that are well-known to those of skill in the art which encode the novel tIC851 polypeptide, peptides, peptide fragments, subunits, or functional domains disclosed herein.

As used herein, the term “nucleic acid sequence” or “polynucleotide” refers to a nucleic acid molecule that has been isolated free of the total genomic DNA or otherwise of a particular species. Therefore, a nucleic acid sequence or polynucleotide encoding an endotoxin polypeptide refers to a nucleic acid molecule that comprises at least a first crystal protein-encoding sequence yet is isolated away from, or purified free from, total genomic DNA of the species from which the nucleic acid sequence is obtained, which in the instant case is the genome of the Gram-positive bacterial genus, Bacillus, and in particular, the species of Bacillus known as B. thuringiensis. Included within the term “nucleic acid sequence”, are polynucleotide sequences and smaller fragments of such sequences, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, virions, baculoviruses, artificial chromosomes, viruses, and the like. Accordingly, polynucleotide sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% nucleic acid sequence identity or functional equivalence to the polynucleotide sequence of SEQ ID NO:7 will be sequences that are “essentially as set forth in SEQ ID NO:7.” Highly preferred sequences are those which are preferably from about 91% to about 100% identical or functionally equivalent to the nucleotide sequence of SEQ ID NO:7. Other preferred sequences that encode tIC851- or tIC851-related sequences are those which are from about 81% to about 90% identical or functionally equivalent to the polynucleotide sequence set forth in SEQ ID NO:7. Likewise, sequences that are from about 71% to about 80% identical or functionally equivalent to the polynucleotide sequence set forth in SEQ ID NO:7 are also contemplated to be useful in the practice of the present invention.

Similarly, a polynucleotide comprising an isolated, purified, or selected gene or sequence region refers to a polynucleotide which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term “gene” is used for simplicity to refer to a functional protein-, or polypeptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, operator sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. In certain embodiments, a nucleic acid segment will comprise at least a first gene that encodes a polypeptide comprising the sequence of SEQ ID NO:8.

To permit expression of the gene, and translation of the mRNA into mature polypeptide, the nucleic acid sequence preferably also comprises at least a first promoter operably linked to the gene to express the insecticidal polypeptide in a host cell transformed with this nucleic acid sequence. The promoter may be an endogenous promoter, or alternatively, a heterologous promoter selected for its ability to promote expression of the gene in one or more particular cell types. For example, in the creation of transgenic plants and plant cells comprising a tIC851 gene, the heterologous promoter of choice is one that is plant-expressible, and in many instances, may preferably be a plant-expressible promoter that is tissue- or cell cycle-specific. The selection of plant-expressible promoters is well-known to those skilled in the art of plant transformation, and exemplary suitable promoters are described herein. In certain embodiments, the plant-expressible promoter may be selected from the group consisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBP carboxylase promoter.

“Isolated substantially away from other coding sequences” means that the gene of interest, in this case, a gene encoding a bacterial crystal protein, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.

It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of SEQ ID NO:8, including the DNA sequence which is particularly disclosed in SEQ ID NO:7. Recombinant vectors and isolated DNA segments may therefore variously include the polypeptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA sequences of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon degeneracy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively). Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA sequence, whether encoding a full-length insecticidal protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein. In many cases, the promoter may be the native tIC851 promoter, or alternatively, a heterologous promoter, such as those of bacterial origin (including promoters from other crystal proteins), fungal origin, viral, phage or phagemid origin (including promoters such as CaMV35, and its derivatives, T3, T7, λ, and φ promoters and the like), or plant origin (including constitutive, inducible, and/or tissue-specific promoters and the like).

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA sequence under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA sequence encoding a crystal protein or peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA sequence, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).

In yet another aspect, the present invention provides methods for producing a transgenic plant that expresses a selected nucleic acid sequence comprising a sequence region that encodes the novel endotoxin polypeptides of the present invention. The process of producing transgenic plants is well-known in the art. In general, the method comprises transforming a suitable plant host cell with a DNA sequence that contains a promoter operatively linked to a coding region that encodes one or more tIC851 polypeptides. Such a coding region is generally operatively linked to at least a first transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the polypeptide in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant crystal protein expressed in a particular transgenic cell, the invention also provides for the expression of crystal protein antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic plants which express a gene, gene sequence, or sequence region that encodes at least one or more of the novel polypeptide compositions disclosed herein. As used herein, the term “transgenic plant” is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (“expressed”), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression.

It is contemplated that in some instances the genome of a transgenic plant of the present invention will have been augmented through the stable introduction of one or more transgenes, either native, synthetically modified, or mutated, that encodes an insecticidal polypeptide that is identical to, or highly homologous to the polypeptide disclosed in SEQ ID NO:8. In some instances, more than one transgene will be incorporated into the genome of the transformed host plant cell. Such is the case when more than one crystal protein-encoding DNA sequence is incorporated into the genome of such a plant. In certain situations, it may be desirable to have one, two, three, four, or even more B. thuringiensis crystal proteins (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic plant. Alternatively, a second transgene may be introduced into the plant cell to confer additional phenotypic traits to the plant. Such transgenes may confer resistance to one or more insects, bacteria, fungi, viruses, nematodes, or other pathogens.

A preferred gene which may be introduced includes, for example, a crystal protein-encoding DNA sequence from bacterial origin, and particularly one or more of those described herein which are obtained from Bacillus spp. Highly preferred nucleic acid sequences are those obtained from B. thuringiensis, or any of those sequences which have been genetically engineered to decrease or increase the insecticidal activity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of plant cells, and regeneration of a transgenic cell line from a transformed cell, cell culture, embryo, or callus tissue are well-known in the art, and are discussed herein. Vectors, (including plasmids, cosmids, phage, phagemids, baculovirus, viruses, virions, BACs [bacterial artificial chromosomes], YACs [yeast artificial chromosomes]) comprising at least a first nucleic acid segment encoding an insecticidal polypeptide for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed crystal proteins. These nucleic acid constructs can further include structures such as promoters, enhancers, polylinkers, introns, terminators, or even gene sequences which have positively- or negatively-regulating activity upon the cloned δ-endotoxin gene as desired. The DNA sequence or gene may encode either a native or modified crystal protein, which will be expressed in the resultant recombinant cells, and/or which will confer to a transgenic plant comprising such a segment, an improved phenotype (in this case, increased resistance to insect attack, infestation, or colonization).

The preparation of a transgenic plant that comprises at least one polynucleotide sequence encoding a tIC851 or tIC851-derived polypeptide for the purpose of increasing or enhancing the resistance of such a plant to attack by a target insect represents an important aspect of the invention. In particular, the inventors describe herein the preparation of insect-resistant monocotyledonous or dicotyledonous plants, by incorporating into such a plant, a transgenic DNA sequence encoding at least one tIC851 polypeptide toxic to a coleopteran insect.

In a related aspect, the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and a seed produced by the progeny of the original transgenic plant, produced in accordance with the above process. Such progeny and seeds will have a crystal protein-encoding transgene stably incorporated into their genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA sequences encoding one or more tIC851 crystal proteins or polypeptides are aspects of this invention. As well-known to those of skill in the art, a progeny of a plant is understood to mean any offspring or any descendant from such a plant.

2.3 DEFINITIONS

The following words and phrases have the meanings set forth below.

A, an: In keeping with long-standing patent tradition, “a” or “an” used throughout this disclosure is intended to mean “one or more.”

Comprising, comprises: In keeping with long-standing patent tradition, “comprising” and “comprises” used throughout this disclosure is intended to mean “including, but not limited to.”

Expression: The combination of intracellular processes, including at least transcription and often the subsequent translation of mRNA of a coding DNA molecule such as a structural gene to produce a polypeptide.

Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene or sequence to be transcribed and to which an RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene or sequence to be transcribed.

Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast or explant).

Structural gene: A DNA sequence that encodes a messenger RNA which can be transcribed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell, protoplast, or organelle within a cell, in which that exogenous DNA is incorporated into DNA native to the cell, or is capable of autonomous replication within the cell.

Transformed cell: A cell whose genotype has been altered by the introduction of an exogenous DNA sequence into that cell.

Transgenic cell: Any cell derived from or regenerated from a transformed cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.

Transgenic plant: A plant or a progeny of any generation of the plant that was derived from a transformed plant cell or protoplast, wherein the plant nucleic acids contains an exogenous selected nucleic acid sequence region not originally present in a native, non-transgenic plant of the same variety. The terms “transgenic plant” and “transformed plant” have sometimes been used in the art as synonymous terms to define a plant whose native DNA has been altered to contain a heterologous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast cells as being a transgenic plant. Preferably, transgenic plants of the present invention include those plants that comprise at least a first selected polynucleotide that encodes an insecticidal polypeptide. This selected polynucleotide is preferably a δ-endotoxin coding region (or gene) operably linked to at least a first promoter that expresses the coding region to produce the insecticidal polypeptide in the transgenic plant. Preferably, the transgenic plants of the present invention that produce the encoded polypeptide demonstrate a phenotype of improved resistance to target insect pests. Such transgenic plants, their progeny, descendants, and seed from any such generation are preferably insect resistant plants.

Vector: A nucleic acid molecule capable of replication in a host cell and/or to which another nucleic acid sequence can be operably linked so as to bring about replication of the attached segment. Plasmids, phage, phagemids, and cosmids are all exemplary vectors. In many embodiments, vectors are used as a vehicle to introduce one or more selected polynucleotides into a host cell, thereby generating a “transformed” or “recombinant” host cell.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 illustrates the nucleotide sequence and amino acid sequence translation of the tIC851 gene as derived from strains EG4135 and 4268.

FIG. 2 illustrates an amino acid sequence alignment of the related proteins CryET70 and Cry22Aa, as well as the bestfit alignment of tIC851.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 4.1 Some Advantages of the Invention

The present invention provides a novel δ-endotoxin, designated tIC851, which is highly toxic to the cotton boll weevil, Anthonotnus grandis Boheman. This protein has an amino acid sequence which is substantially unrelated to other δ-endotoxins that are toxic to coleopteran insects. The identification of Cry22Aa and CryET70 represented a new class of insecticidal crystal proteins. Unlike other WCRW toxic insecticidal crystal proteins from B. thuringiensis, CryET70 does not have significant toxicity to SCRW or CPB. The only known protein that is related to CryET70 is Cry22Aa, an insecticidal crystal protein that is reported to be toxic only to hymenopteran insects (GenBank Accession No. 134547). The inventors herein disclose a novel Bacillus thuringiensis δ-endotoxin displaying only insubstantial similarity to either CryET70 or to Cry22Aa, and displaying substantial differences in insecticidal spectrum and activity when compared to both of these proteins. The inventors also disclose that both CryET70 and Cry22Aa have significant toxicity to larvae of the cotton boll weevil.

4.2 Insect Pests

Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests. Particularly problematic coleopteran pests are identified in Table 1.

TABLE 1 TAXONOMY OF COLEOPTERAN PESTS IN THE SUBORDERS ARCHOSTEMATA AND POLYPHAGA Family Subfamily Tribe Genus Species Infraorder &/or Superfamily Cupedidae (reticulated Priacma P. serrata beetles) Bostrichiformia Dermestidae (skin and Attagenus A. pellio larder beetles) Chrysomeliformia Cerambycidae (long- Agapanthia Agapanthia sp. horned beetles) Lepturinae Leptura Leptura sp. (flower long-horned beetle) Rhagium Rhagium sp. Megacyllene M. robiniae Prioninae Derobrachus D. geminatus Tetraopes T. tetropthalmus Chrysomelidae (leaf Chlamisinae Exema E. neglecta beetles) Chrysomelinae Chrysomelini Chrysomela C. tremula, Chrysomela sp. Oreina O. cacaliae Doryphorini Chrysoline Chrysolina sp. Leptinotarsa L. decemlineata (Colorado potato beetle) Infraorder Gonioctenini Gonioctena G. fornicata, G. holdausi, G. intermedia, G. interposita, G. kamikawai, G. linnaeana, G. nigroplagiata, G. occidentalis, G. olivacea, G. pallida, G. quin- quepunctata, G. rubripennis, G. rufipes, G. tredecim-maculata, G. variabilis, G. viminalis Timarchini Timarcha Timarcha sp. Criocerinae Oulema Oulema sp. Galerucinae Galerucini Monoxia M. inornata, Monoxia sp. Ophraella O. arctica, O. artemisiae, O. bilineata, O. communa, O. conferta, O. cribrata, O. notata, O. notulata, O. nuda, O. pilosa, O. sexvittata, O. slobodkini Luperini Cerotoma C. trifurcata Diabrotica D. barberi (northern corn rootworm), D. undecimpunctata, (southern corn rootworm), D. virgifera (western corn rootworm) unclassified Lachnaia Lachnaia sp. Chrysomelidae Epitrix E. cucumeris (Harris) (potato flea beetle), E. fuscala (eggplant flea beetle) Curculionidae (weevils) Curculioninae Anthonomus A. grandis (boll weevil) Entiminae Naupactini Aramigus A. conirostris, A. globoculus, A. intermedius, A. planioculus, A. tesselatus Otiorhynchus Otiorhynchus sp. Phyllobiini Diaprepes D. abbreviata Phyllobius Phyllobius sp. Galapaganus G. galapagoensis Hyperinae Hypera H. brunneipennis (Egyptian alfalfa weevil), H. postica (alfalfa weevil), H. punctata (clover leaf weevil) Molytinae Pissodes P. affinis, P. nemorensis, P. schwarzi, P. strobi, P. terminalis Rhynchophorinae Sitophilini Sitophilus S. granarius (granary weevil), S. zeamais (maize weevil) Nemonychidae Lebanorhinus L. succinus Scolytidae Ips I. acuminatus, I. amitinus, I. cembrae, I. duplicatus, I. mannsfeldi, I. sexdentatus, I. typographus Orthotomicus O. erosus Tomicus T. minor Cucujiformia Coccinellidae (ladybird Epilachna E. borealis (squash ladybird beetle), beetles) E. varivstis (Mexican bean beetle) Cucujidae (flat bark Cryptolestes C. ferrugineus beetles) Oryzaephilus O. surinamensis (saw-toothed grain (grain beetles) beetle) Lagriidae (long-joined Lagria Lagria sp. beetles) Meloidae (blister beetles) Epicauta E. funebris Meloe M. proscarabaeus Rhipiphoridae Rhipiphorus R. fasciatus Tenebrionidae (darkling Alphitobius A. diaperinus ground beetles) (lesser mealworm) Hegeter H. amaroides, H. brevicollis, H. costipennis, H. fernandezi, H. glaber, H. gomerensis, H. gran-canariensis, H. impressus, H. intercedens, H. lateralis, H. plicifrons, H. politus, H. subrotundatus, H. tenui-punctatus, H. transversus, H. webbianus Misolampus M. goudoti Palorus P. ficicola, P. ratzeburgi (small-eyed flour beetle), P. subdepressus (depressed flour beetle) Pimelia P. baetica, P. canariensis, P. criba, P. elevata, P. estevezi, P. fernan-dezlopezi, P. grandis, P. granulicollis, P. integra, P. interjecta, P. laevigata, P. lutaria, P. radula, P. sparsa, P. variolosa Tenebrio T. molitor (yellow mealworm), T. obscurus (dark mealworm) Tentyria T. schaumi Tribolium T. brevicornis, T. castaneum (red flour beetle), T. confusum (confused flour beetle), T. freemani, T. madens Zophobas Z. atratus Z. rugipes Elateriformia - Octinodes Octinodes sp. Superfamily Elateroidea Pyrophorus P. plagio-phthalamus Scarabaeiformia Lucanidae (Stag beetles) Dorcus D. parallelo-pipedus Lucanus L. cervus Scarabaeidae (lamellicorn Allomyrina A. dichotoma beetles) Cetoniinae (flower Pachnoda P. marginata beetle) Dynastinae Xyloryctes X. faunus Geotrupinae (earth- Geotrupes G. stercorosus boring dung beetles) Melonlonthinae Costelytra C. zealandica (chafers) Holotrichia H. diomphalia Melolontha M. melolontha (cockchafer) Odontria O. striata O. variegata Prodontria P. bicolorata, P. capito, P. lewisi, P. tarsis, P. modesta, P. pinguis, P. praelatella, P. truncata, Prodontria sp. Scythrodes S. squalidus Rutelinae (shining Popillia P. japonica (Japanese beetle) leaf chafers) Scarabaeinae Copris C. lunaris (black dung beetle) Scarabaeus Scarabaeus sp. (scarab) Staphyliniformia Hydrophilidae Cercyon Cercyon sp. Silphidae Nicrophorus N. americanus, N. marginatus, N. orbicollis, N. tomentosus Staphylinidae (rove Carpelimus Carpelimus sp. beetles) Quedius Q. mesomelinus Tachyporus Tachyporus sp. Xantholinus Xantholinus sp.

4.3 Probes and Primers

In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected crystal protein-encoding gene sequence, e.g., a sequence such as that shown in SEQ ID NO:8 (tIC851), SEQ ID NO:10 (Cry22Aa), and SEQ ID NO:2 (CryET70). The ability of such DNAs and nucleic acid probes to specifically hybridize to a crystal protein-encoding gene sequence lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of a crystal protein gene from B. thuringiensis using thermal amplification technology. Sequences of related crystal protein genes from other species may also be amplified using such primers.

To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least an about 23 to about 40 or so long nucleotide stretch of a crystal protein-encoding sequence, such as that shown in SEQ ID NO:7 (tIC851), SEQ ID NO:9 (cry22Aa), or SEQ ID NO:1 (cryET70). A size of at least about 14 or 15 or so nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than about 23 or so bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of about 14 to about 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195, and 4,683,202, specifically incorporated herein by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.

4.4 Expression Vectors

The present invention contemplates a polynucleotide of the present invention comprised within one or more expression vectors. Thus, in one embodiment an expression vector comprises a nucleic acid segment containing a tIC851 gene operably linked to a promoter which expresses the gene. Additionally, the coding region may also be operably linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region, and the transcription-terminating region halts transcription at some point 3′ of the coding region.

As used herein, the term “operatively linked” means that a promoter is connected to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.

In a preferred embodiment, the recombinant expression of DNAs encoding the crystal proteins of the present invention is preferable in a Bacillus host cell. Preferred host cells include B. thuringiensis, B. megaterium, B. subtilis, and related bacilli, with B. thuringiensis host cells being highly preferred. Promoters that function in bacteria are well-known in the art. An exemplary and preferred promoter for the Bacillus-derived crystal proteins include any of the known crystal protein gene promoters, including the tIC851 gene promoter itself. Alternatively, mutagenized or recombinant promoters may be engineered by the hand of man and used to promote expression of the novel gene segments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encoding the crystal proteins of the present invention is performed using a transformed Gram-negative bacterium such as an E. coli or Pseudomonas spp. host cell. Promoters which function in high-level expression of target polypeptides in E. coli and other Gram-negative host cells are also well-known in the art.

Where an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants. Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).

A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near-constitutive, such as the CaMV 35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S, increases in polypeptide expression are found in a variety of transformed plant tissues (e.g., callus, leaf, seed and root). Alternatively, the effects of transformation can be directed to specific plant tissues by using plant integrating vectors containing a tissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The Lectin protein in soybean seeds is encoded by a single gene (Le1) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed storage protein specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes a polypeptide of interest is engineered to be under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991a). The expression of the polypeptide is directed specifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35S transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et al., 1985). pCaMVCN (available from Pharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV 35S promoter.

In preferred embodiments, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. One preferred drug resistance marker is the gene whose expression results in kanamycin resistance; i.e., the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II (nptII) and nopaline synthase 3′ non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are specifically incorporated herein by reference in their entirety. Those vectors can be modified to include a coding sequence in accordance with the present invention.

A variety of methods have been developed to operatively insert a DNA sequence into a vector via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA sequence to be inserted and to the vector DNA. The vector and DNA sequence are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to confer insecticidal activity to a cell is preferably a tIC851 B. thuringiensis crystal protein-encoding gene. In preferred embodiments, such a polypeptide has the amino acid residue sequence of SEQ ID NO:8, or a functional equivalent thereof. In accordance with such embodiments, a coding region comprising the DNA sequence of SEQ ID NO:7 is also preferred.

4.5 Characteristic of the tIC851 Polypeptide Isolated from EG4135

The present invention provides a novel polypeptide that defines a whole or a portion of a B. thuringiensis tIC851 crystal protein.

In a preferred embodiment, the invention discloses and claims an isolated and purified tIC851 protein. The tIC851 protein isolated from EG4135 comprises a 632 amino acid sequence, and has a calculated molecular mass of approximately 69,527 Da. tIC851 has a calculated isoelectric constant (pI) equal to 5.80. The amino acid composition of the tIC851 protein is given in Table 2.

TABLE 2 AMINO ACID COMPOSITION OF tIC851 Amino Acid # Residues % Total Ala 45 7.1 Arg 13 2.1 Asn 40 6.3 Asp 49 7.8 Cys 1 0.2 Gln 13 2.1 Glu 41 6.5 Gly 47 7.4 His 12 1.9 Ile 62 9.8 Leu 29 4.6 Lys 51 8.1 Met 5 0.8 Phe 22 3.5 Pro 34 5.4 Ser 34 5.4 Thr 57 9.0 Tro 8 1.3 Tyr 25 3.9 Val 44 6.9 90 14 64 10 55 9 195 31 Acidic (Asp + Glu) Basic (Arg + Lys) Aromatic (Phe + Trp + Tyr) Hydrophobic (Aromatic + Ile + Leu + Met + Val)

4.6 Nomenclature of the Novel Proteins

The inventors have arbitrarily assigned the designation tIC851 to the novel protein of the invention. Likewise, the arbitrary designation of tIC851 has been assigned to the novel nucleic acid sequence which encodes this polypeptide. Formal assignment of gene and protein designations based on the revised nomenclature of crystal protein endotoxins will be assigned by a committee on the nomenclature of B. thuringiensis, formed to systematically classify B. thuringiensis crystal proteins. The inventors contemplate that the arbitrarily assigned designations of the present invention will be superseded by the official nomenclature assigned to these sequences, and that based on the lack of identity or substantial similarity to other known insecticidal protein isolated from Bacillus thuringiensis, the tIC851 protein will be alone in a separate category and class of proteins.

4.7 Transformed Host Cells and Transgenic Plants

Methods and compositions for transforming a bacterium, a yeast cell, a plant cell, or an entire plant with one or more expression vectors comprising a crystal protein-encoding gene sequence are further aspects of this disclosure. A transgenic bacterium, yeast cell, plant cell or plant derived from such a transformation process or the progeny and seeds from such a transgenic plant are also further embodiments of the invention.

Means for transforming bacteria and yeast cells are well known in the art. Typically, means of transformation are similar to those well known means used to transform other bacteria or yeast such as E. coli or Saccharomyces cerevisiae. Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain. Suitable methods for introducing transforming DNA into a cell consist of but are not limited to Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).

4.7.1 Microprojectile Bombardment

A particularly advantageous method for delivering transforming DNA sequences into plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

4.7.2 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).

Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

4.7.3 Gene Expression in Plants

To overcome limitations in foreign gene expression in plants, particular sequences and signals in RNAs that have the potential for having a specific effect on RNA stability have been identified. In certain embodiments of the invention, therefore, there is a desire to optimize expression of the disclosed nucleic acid segments in planta. One particular method of doing so, is by alteration of the bacterial gene to remove sequences or motifs which decrease expression in a transformed plant cell. The process of engineering a coding sequence for optimal expression in planta is often referred to as “plantizing” a DNA sequence.

Particularly problematic sequences are those which are A+T rich. Unfortunately, since B. thuringiensis has an A+T rich genome, native crystal protein gene sequences must often be modified for optimal expression in a plant. The sequence motif ATTTA (or AUUUA as it appears in RNA) has been implicated as a destabilizing sequence in mammalian cell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich 3′ untranslated regions, and these regions often have the ATTTA sequence, sometimes present in multiple copies or as multimers (e.g., ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ end of an unstable mRNA to a stable RNA (globin or VA1) decreased the stable RNA's half life dramatically. They further showed that a pentamer of ATTTA had a profound destabilizing effect on a stable message, and that this signal could exert its effect whether it was located at the 3′ end or within the coding sequence. However, the number of ATTTA sequences and/or the sequence context in which they occur also appear to be important in determining whether they function as destabilizing sequences. Shaw and Kamen showed that a trimer of ATTTA had much less effect than a pentamer on mRNA stability and a dimer or a monomer had no effect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTA such as a pentamer automatically create an A+T rich region. This was shown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs, the ATTTA sequence may be present in only a single copy, but it is often contained in an A+T rich region. From the animal cell data collected to date, it appears that ATTTA at least in some contexts is important in stability, but it is not yet possible to predict which occurrences of AMA are destabilizing elements or whether any of these effects are likely to be seen in plants. Table 3 lists some of the more common AT rich sequences identified as problematic when present in a coding sequence for which high levels of expression are desired.

The addition of a polyadenylate string to the 3′ end is common to most eukaryotic mRNAs, both plant and animal. The currently accepted view of polyA addition is that the nascent transcript extends beyond the mature 3′ terminus. Contained within this transcript are signals for polyadenylation and proper 3′ end formation. This processing at the 3′ end involves cleavage of the mRNA and addition of polyA to the mature 3′ end. By searching for consensus sequences near the polyA tract in both plant and animal mRNAs, it has been possible to identify consensus sequences that apparently are involved in polyA addition and 3′ end cleavage. The same consensus sequences seem to be important to both of these processes. These signals are typically a variation on the sequence AATAAA. In animal cells, some variants of this sequence that are functional have been identified; in plant cells there seems to be an extended range of functional sequences (Wickens and Stephenson, 1984; Dean et al., 1986). Because all of these consensus sequences are variations on AATAAA, they all are A+T rich sequences.

TABLE 3 POLYADENYLATION SITES IN PLANT GENES PA AATAAA Major consensus site P1A AATAAT Major plant site P2A AACCAA Minor plant site P3A ATATAA ″ P4A AATCAA ″ P5A ATACTA ″ P6A ATAAAA ″ P7A ATGAAA ″ P8A AAGCAT ″ P9A ATTAAT ″ P10A ATACAT ″ P11A AAAATA ″ P12A ATTAAA Minor animal site P13A AATTAA ″ P14A AATACA ″ P15A CATAAA ″

The present invention provides a method for preparing synthetic plant genes which genes express their protein product at levels significantly higher than the wild-type genes which were commonly employed in plant transformation heretofore. In another aspect, the present invention also provides novel synthetic plant genes which encode non-plant proteins.

As described above, the expression of native B. thuringiensis genes in plants is often problematic. The nature of the coding sequences of B. thuringiensis genes distinguishes them from plant genes as well as many other heterologous genes expressed in plants. In particular, B. thuringiensis genes are very rich (−62%) in adenine (A) and thymine (T) while plant genes and most other bacterial genes which have been expressed in plants are on the order of 45-55% A+T.

Due to the degeneracy of the genetic code and the limited number of codon choices for any amino acid, most of the “excess” A+T of the structural coding sequences of some Bacillus species are found in the third position of the codons. That is, genes of some Bacillus species have A or T as the third nucleotide in many codons. Thus A+T content in part can determine codon usage bias. In addition, it is clear that genes evolve for maximum function in the organism in which they evolve. This means that particular nucleotide sequences found in a gene from one organism, where they may play no role except to code for a particular stretch of amino acids, have the potential to be recognized as gene control elements in another organism (such as transcriptional promoters or terminators, polyA addition sites, intron splice sites, or specific mRNA degradation signals). It is perhaps surprising that such misread signals are not a more common feature of heterologous gene expression, but this can be explained in part by the relatively homogeneous A+T content (˜50%) of many organisms. This A+T content plus the nature of the genetic code put clear constraints on the likelihood of occurrence of any particular oligonucleotide sequence. Thus, a gene from E. coli with a 50% A+T content is much less likely to contain any particular A+T rich segment than a gene from B. thuringiensis.

Typically, to obtain high-level expression of the δ-endotoxin genes in plants, existing structural coding sequence (“structural gene”) which codes for the δ-endotoxin are modified by removal of ATTTA sequences and putative polyadenylation signals by site directed mutagenesis of the DNA comprising the structural gene. It is most preferred that substantially all the polyadenylation signals and ATTTA sequences are removed although enhanced expression levels are observed with only partial removal of either of the above identified sequences. Alternately if a synthetic gene is prepared which codes for the expression of the subject protein, codons are selected to avoid the ATTTA sequence and putative polyadenylation signals. For purposes of the present invention putative polyadenylation signals include, but are not necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. In replacing the ATTTA sequences and polyadenylation signals, codons are preferably utilized which avoid the codons which are rarely found in plant genomes.

The selected DNA sequence is scanned to identify regions with greater than four consecutive adenine (A) or thymine (T) nucleotides. The A+T regions are scanned for potential plant polyadenylation signals. Although the absence of five or more consecutive A or T nucleotides eliminates most plant polyadenylation signals, if there are more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then the nucleotide sequence of this region is preferably altered to remove these signals while maintaining the original encoded amino acid sequence.

The second step is to consider the about 15 to about 30 or so nucleotide residues surrounding the A+T rich region identified in step one. If the A+T content of the surrounding region is less than 80%, the region should be examined for polyadenylation signals. Alteration of the region based on polyadenylation signals is dependent upon (1) the number of polyadenylation signals present and (2) presence of a major plant polyadenylation signal.

The extended region is examined for the presence of plant polyadenylation signals. The polyadenylation signals are removed by site-directed mutagenesis of the DNA sequence. The extended region is also examined for multiple copies of the ATTTA sequence which are also removed by mutagenesis.

It is also preferred that regions comprising many consecutive A+T bases or G+C bases are disrupted since these regions are predicted to have a higher likelihood to form hairpin structure due to self-complementarity. Therefore, insertion of heterogeneous base pairs would reduce the likelihood of self-complementary secondary structure formation which are known to inhibit transcription and/or translation in some organisms. In most cases, the adverse effects may be minimized by using sequences which do not contain more than five consecutive A+T or G+C.

4.7.4 Synthetic Oligonucleotides for Mutagenesis

When oligonucleotides are used in the mutagenesis, it is desirable to maintain the proper amino acid sequence and reading frame, without introducing common restriction sites such as BglII, HindIII, SacI, KpnI, EcoRI, NcoI, PstI and SalI into the modified gene. These restriction sites are found in poly-linker insertion sites of many cloning vectors. Of course, the introduction of new polyadenylation signals, ATTTA sequences or consecutive stretches of more than five A+T or G+C, should also be avoided. The preferred size for the oligonucleotides is about 40 to about 50 bases, but fragments ranging from about 18 to about 100 bases have been utilized. In most cases, a minimum of about 5 to about 8 base pairs of homology to the template DNA on both ends of the synthesized fragment are maintained to insure proper hybridization of the primer to the template. The oligonucleotides should avoid sequences longer than five base pairs A+T or G+C. Codons used in the replacement of wild-type codons should preferably avoid the TA or CG doublet wherever possible. Codons are selected from a plant preferred codon table (such as Table 4 below) so as to avoid codons which are rarely found in plant genomes, and efforts should be made to select codons to preferably adjust the G+C content to about 50%.

TABLE 4 PREFERRED CODON USAGE IN PLANTS Amino Acid Codon Percent Usage in Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29 AGG 23 LEU CUA 8 CUC 20 CUG 10 CUU 28 UUA 5 UUG 30 SER UCA 14 UCC 26 UCG 3 UCU 21 AGC 21 AGU 15 THR ACA 21 ACC 41 ACG 7 ACU 31 PRO CCA 45 CCC 19 CCG 9 CCU 26 ALA GCA 23 GCC 32 GCG 3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45 AUU 43 VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU 28 GLN CAA 64 CAG 36 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU 52 TYR UAC 68 UAU 32 CYS UGC 78 UGU 22 PHE UUC 56 UUU 44 MET AUG 100 TRP UGG 100

Regions with many consecutive A+T bases or G+C bases are predicted to have a higher likelihood to form hairpin structures due to self-complementarity. Disruption of these regions by the insertion of heterogeneous base pairs is preferred and should reduce the likelihood of the formation of self-complementary secondary structures such as hairpins which are known in some organisms to inhibit transcription (transcriptional terminators) and translation (attenuators).

Alternatively, a completely synthetic gene for a given amino acid sequence can be prepared, with regions of five or more consecutive A+T or G+C nucleotides being avoided. Codons are selected avoiding the TA and CG doublets in codons whenever possible. Codon usage can be normalized against a plant preferred codon usage table (such as Table 4) and the G+C content preferably adjusted to about 50%. The resulting sequence should be examined to ensure that there are minimal putative plant polyadenylation signals and AMA sequences. Restriction sites found in commonly used cloning vectors are also preferably avoided. However, placement of several unique restriction sites throughout the gene is useful for analysis of gene expression or construction of gene variants.

4.8 Methods for Producing Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with a recombinant tIC851 gene sequence, the expression of the encoded crystal protein (i.e. a bacterial crystal protein or polypeptide having insecticidal activity against Coleopterans) can result in the formation of insect-resistant plants.

A transgenic plant of this invention thus has an increased amount of a coding region (e.g., a gene) that encodes a polypeptide in accordance with SEQ ID NO:8. A preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny. A more preferred transgenic plant is homozygous for that gene, and transmits that gene to all of its offspring upon sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for, by way of example, increased insecticidal capacity against coleopteran insects, preferably in the field, under a range of environmental conditions.

Transgenic plants comprising one or more transgenes that encode a polypeptide in accordance with SEQ ID NO:8 will preferably exhibit a phenotype of improved or enhanced insect resistance to the target coleopteran insects as described herein. These plants will preferably provide transgenic seeds, which will be used to create lineages of transgenic plants (i.e. progeny or advanced generations of the original transgenic plant) that may be used to produce seed, or used as animal or human foodstuffs, or to produce fibers, oil, fruit, grains, or other commercially-important plant products or plant-derived components. In such instances, the progeny and seed obtained from any generation of the transformed plants will contain the selected and stably integrated transgene that encodes the δ-endotoxin of the present invention. The transgenic plants of the present invention may be crossed to produce hybrid or inbred lines with one or more plants that have desirable properties. In certain circumstances, it may also be desirable to create transgenic plants, seed, and progeny that contain one or more additional transgenes incorporated into their genome in addition to the transgene encoding the polypeptide of the invention. For example, the transgenic plants may contain a second gene encoding the same, or a different insect-resistance polypeptide, or alternatively, the plants may comprise one or more additional transgenes such as those conferring herbicide resistance, fungal resistance, bacterial resistance, stress, salt, or drought tolerance, improved stalk or root lodging, increased starch, grain, oil, carbohydrate, amino acid, protein production, and the like.

4.9 Isolating Homologous Gene and Gene Fragments

The genes and δ-endotoxins according to the subject invention include not only the full length sequences disclosed herein but also fragments of these sequences, or fusion proteins, which retain the characteristic insecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person skill in this art that insecticidal δ-endotoxins can be identified and obtained through several means. The specific genes, or portions thereof, may be obtained from a culture depository, or constructed synthetically, for example, by use of a gene machine. Variations of these genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. Also, genes which code for active fragments may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these δ-endotoxins.

Equivalent δ-endotoxins and/or genes encoding these equivalent δ-endotoxins can also be isolated from Bacillus strains and/or DNA libraries using the teachings provided herein. For example, antibodies to the δ-endotoxins disclosed and claimed herein can be used to identify and isolate other δ-endotoxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the δ-endotoxins which are most constant and most distinct from other B. thuringiensis δ-endotoxins. These antibodies can then be used to specifically identify equivalent δ-endotoxins with the characteristic insecticidal activity by immunoprecipitation, enzyme linked immunoassay (ELISA), or Western blotting.

A further method for identifying the δ-endotoxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a detectable label. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probes detectable label provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying formicidal δ-endotoxin genes of the subject invention.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, by methods currently known to an ordinarily skilled artisan, and perhaps by other methods which may become known in the future.

The potential variations in the probes listed is due, in part, to the redundancy of the genetic code. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins. Therefore different nucleotide sequences can code for a particular amino acid. Thus, the amino acid sequences of the B. thuringiensis δ-endotoxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a δ-endotoxin encoding a gene of the invention. Such mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art.

4.10 Recombinant Host Cells

The nucleotide sequences of the subject invention may be introduced into a wide variety of microbial and eukaryotic hosts. As hosts for recombinant expression of tIC851 polypeptides, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and Gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the genetic constructs of the present invention into the host cell, availability of expression systems, efficiency of expression, stability of the gene of interest in the host, and the presence of auxiliary genetic capabilities.

A large number of microorganisms known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops may also be desirable host cells for manipulation, propagation, storage, delivery and/or mutagenesis of the disclosed genetic constructs. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Bacillus (including the species and subspecies B. thuringiensis kurstaki HD-1, B. thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensis berliner, B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B. thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensis galleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B. thuringiensis entomocidus, B. thuringiensis tenebrionis and B. thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing a selected genetic construct into the host, availability of expression systems, efficiency of expression, stability of the polynucleotide in the host, and the presence of auxiliary genetic capabilities. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

4.11 Polynucleotide Sequences

DNA compositions encoding the insecticidally-active polypeptides of the present invention are particularly preferred for delivery to recipient plant cells, and ultimately in the production of insect-resistant transgenic plants. For example, DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the DNA element to be expressed in the plant cell, and the like, may be employed.

4.12 Methods for Preparing Mutagenized Polynucleotide Sequences

In certain circumstances, it may be desirable to modify or alter one or more nucleotides in one or more of the polynucleotide sequences disclosed herein for the purpose of altering or changing the insecticidal activity or insecticidal specificity of the encoded polypeptide. In general, the means and methods for mutagenizing a DNA sequences are well-known to those of skill in the art. Modifications to such sequences may be made by random, or site-specific mutagenesis procedures. The polynucleotides may be modified by the addition, deletion, or substitution of one or more nucleotides from the sequence encoding the insecticidally-active polypeptide.

Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular region. In particular, site-specific mutagenesis is a technique useful in the preparation of mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

The preparation of sequence variants of the selected δ-endotoxin-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of DNA sequences may be obtained. For example, recombinant vectors encoding the desired sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesis procedure” refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term “oligonucleotide directed mutagenesis procedure” also is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.

A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5′-[α-thio]triphosphates in one strand of a restriction site (Walker et al., 1992, incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.

4.13 Post-Transcriptional Events Affecting Expression of Transgenes in Plants

In many instances, the level of transcription of a particular transgene in a given host cell is not always indicative of the amount of protein being produced in the transformed host cell. This is often due to post-transcriptional processes, such as splicing, polyadenylation, appropriate translation initiation, and RNA stability, that affect the ability of a transcript to produce protein. Such factors may also affect the stability and amount of mRNA produced from the given transgene. As such, it is often desirable to alter the post-translational events through particular molecular biology techniques. The inventors contemplate that in certain instances it may be desirable to alter the transcription and/or expression of the polypeptide-encoding nucleic acid constructs of the present invention to increase, decrease, or otherwise regulate or control these constructs in particular host cells and/or transgenic plants.

4.13.1 Efficient Initiation of Protein Translation

The 5′-untranslated leader (5′-UTL) sequence of eukaryotic mRNA plays a major role in translational efficiency. Many early chimeric transgenes using a viral promoter used an arbitrary length of viral sequence after the transcription initiation site and fused this to the AUG of the coding region. More recently studies have shown that the 5′-UTL sequence and the sequences directly surrounding the AUG can have a large effect in translational efficiency in host cells and particularly certain plant species and that this effect can be different depending on the particular cells or tissues in which the message is expressed.

In most eukaryotic mRNAs, the point of translational initiation occurs at the AUG codon closest to the 5″ cap of the transcript. Comparison of plant mRNA sequences and site directed mutagenesis experiments have demonstrated the existence of a consensus sequence surrounding the initiation codon in plants, 5′-UAAACAAUGGCU-3′ (SEQ ID NO:4) (Joshi, 1987; Lutcke et al., 1987). However, consensus sequences will be apparent amongst individual plant species. For example, a compilation of sequences surrounding the initiation codon from 85 maize genes yields a consensus of 5′-(C/G)AUGGCG-3′ (Luehrsen et al., 1994). In tobacco protoplasts, transgenes encoding β-glucuronidase (GUS) and bacterial chitinase showed a 4-fold and an 8-fold increase in expression, respectively, when the native sequences of these genes were changed to encode 5′-ACCAUGG-3′ (Gallie et al., 1987b; Jones et al., 1988). Interestingly, B. thuringiensis has chosen to utilize an alternative initiation codon for the native gene encoding tIC851. The inventors find, as described below, that this codon, although not generally known to encode for other than leucine, is believed to code for methionine in the first position of the tIC851 polypeptide toxin as judged by N-terminal amino acid sequence analysis of the purified toxin. Therefore, for efficiency inplanta, it is intended that the more frequently utilized ATG initiation codon will be used instead.

When producing chimeric transgenes (i.e. transgenes comprising DNA segments from different sources operably linked together), often the 5′-UTL of plant viruses are used. The alfalfa mosaic virus (AMV) coat protein and brome mosaic virus (BMV) coat protein 5′-UTLs have been shown to enhance mRNA translation 8-fold in electroporated tobacco protoplasts (Gallie et al., 1987a; 1987b). A 67-nucleotide derivative (Ω) of the 5′-UTL of tobacco mosaic virus RNA (TMV) fused to the chloramphenicol acetyltransferase (CAT) gene and GUS gene has been shown to enhance translation of reporter genes in vitro (Gallie et al., 1987a; 1987b; Sleat et al., 1987; Sleat et al., 1988). Electroporation of tobacco mesophyll protoplasts with transcripts containing the TMV leader fused to reporter genes CAT, GUS, and LUC produced a 33-, 21-, and 36-fold level of enhancement, respectively (Gallie et al., 1987a; 1987b; Gallie et al., 1991). Also in tobacco, an 83-nt 5′-UTL of potato virus X RNA was shown to enhance expression of the neomycin phosphotransferese II (NptII) 4-fold (Poogin and Skryabin, 1992).

The effect of a 5′-UTL may be different depending on the plant, particularly between dicots and monocots. The TMV 5′-UTL has been shown to be more effective in tobacco protoplasts (Gallie et al., 1989) than in maize protoplasts (Gallie and Young, 1994). Also, the 5′-UTLs from TMV-Ω (Gallie et al., 1988), AMV-coat (Gehrke et al., 1983; Jobling and Gehrke, 1987), TMV-coat (Goelet et al., 1982), and BMV-coat (French et al., 1986) worked poorly in maize and inhibited expression of a luciferase gene in maize relative to its native leader (Koziel et al., 1996). However, the 5′-UTLs from the cauliflower mosaic virus (CaMV) 35S transcript and the maize genes glutelin (Boronat et al., 1986), PEP-carboxylase (Hudspeth and Grula, 1989) and ribulose biphosphate carboxylase showed a considerable increase in expression of the luciferase gene in maize relative to its native leader (Koziel et al., 1996).

These 5′-UTLs had different effects in tobacco. In contrast to maize, the TMV Ω 5′-UTL and the AMV coat protein 5′-UTL enhanced expression in tobacco, whereas the glutelin, maize PEP-carboxylase and maize ribulose-1,5-bisphosphate carboxylase 5′-UTLs did not show enhancement relative to the native luciferase 5′-UTL (Koziel et al., 1996). Only the CaMV 35S 5′-UTL enhanced luciferase expression in both maize and tobacco (Koziel et al., 1996). Furthermore, the TMV and BMV coat protein 5′-UTLs were inhibitory in both maize and tobacco protoplasts (Koziel et al., 1996).

4.13.2 Use of Introns to Increase Expression

Including one or more introns in the transcribed portion of a gene has been found to increase heterologous gene expression in a variety of plant systems (Callis et al., 1987; Maas et al., 1991; Mascerenhas et al., 1990; McElroy et al., 1990; Vasil et al., 1989), although not all introns produce a stimulatory effect and the degree of stimulation varies. The enhancing effect of introns appears to be more apparent in monocots than in dicots. Tanaka et al., (1990) has shown that use of the catalase intron 1 isolated from castor beans increases gene expression in rice. Likewise, the first intron of the alcohol dehydrogenase 1 (Adh1) has been shown to increase expression of a genomic clone of Adh1 comprising the endogenous promoter in transformed maize cells (Callis et al., 1987; Dennis et al., 1984). Other introns that are also able to increase expression of transgenes which contain them include the introns 2 and 6 of Adh1 (Luehrsen and Walbot, 1991), the catalase intron (Tanaka et al., 1990), intron 1 of the maize bronze 1 gene (Callis et al., 1987), the maize sucrose synthase intron 1 (Vasil et al., 1989), intron 3 of the rice actin gene (Luehrsen and Walbot, 1991), rice actin intron 1 (McElroy et al., 1990), and the maize ubiquitin exon 1 (Christensen et al., 1992).

Generally, to achieve optimal expression, the selected intron(s) should be present in the 5′ transcriptional unit in the correct orientation with respect to the splice junction sequences (Callis et al., 1987; Maas et al., 1991; Mascerenhas et al., 1990; Oard et al., 1989; Tanaka et al., 1990; Vasil et al., 1989). Intron 9 of Adh1 has been shown to increase expression of a heterologous gene when placed 3′ (or downstream of) the gene of interest (Callis et al., 1987).

4.13.3 Use of Synthetic Genes to Increase Expression of Heterologous Genes in Plants

When introducing a prokaryotic gene into a eukaryotic host, or when expressing a eukaryotic gene in a non-native host, the sequence of the gene must often be altered or modified to allow efficient translation of the transcript(s) derived form the gene. Significant experience in using synthetic genes to increase expression of a desired protein has been achieved in the expression of Bacillus thuringiensis in plants. Native B. thuringiensis genes are often expressed only at low levels in dicots and sometimes not at all in many species of monocots (Koziel et al., 1996). Codon usage in the native genes is considerably different from that found in typical plant genes, which have a higher G+C content. Strategies to increase expression of these genes in plants generally alter the overall G+C content of the genes. For example, synthetic B. thuringiensis crystal-protein encoding genes have resulted in significant improvements in expression of these endotoxins in various crops including cotton (Perlak et al., 1990; Wilson et al., 1992), tomato (Perlak et al., 1991), potato (Perlak et al., 1993), rice (Cheng et al., 1998), and maize (Koziel et al., 1993).

In a similar fashion the inventors contemplate that the genetic constructs of the present invention, because they contain one or more genes of bacterial origin, may in certain circumstances be altered to increase the expression of these prokaryotic-derived genes in particular eukaryotic host cells and/or transgenic plants which comprise such constructs. Using molecular biology techniques which are well-known to those of skill in the art, one may alter the coding or non coding sequences of the particular tIC851-encoding gene sequences to optimize or facilitate its expression in transformed plant cells at levels suitable for preventing or reducing insect infestation or attack in such transgenic plants.

4.13.4 Use of Promoters in Expression Vectors

The expression of a gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from the coding strand of the DNA by an RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. Transcription of DNA into mRNA is regulated by a region of DNA referred to as the “promoter”. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA. The particular promoter selected should be capable of causing sufficient expression of the coding sequence to result in the production of an effective insecticidal amount of the B. thuringiensis protein.

A promoter is selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region, to ensure sufficient expression of the enzyme coding sequence to result in the production of insecticidal amounts of the B. thuringiensis protein. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near-constitutive (i.e. they drive transcription of the transgene in all tissue), such as the CaMV35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots. Where the promoter is a near-constitutive promoter such as CaMV35S or FMV35S, increases in polypeptide expression are found in a variety of transformed plant tissues and most plant organs (e.g., callus, leaf, seed and root). Enhanced or duplicate versions of the CaMV35S and FMV35S promoters are particularly useful in the practice of this invention (Kay et al., 1987; Rogers, U.S. Pat. No. 5,378,619).

Those skilled in the art will recognize that there are a number of promoters which are active in plant cells, and have been described in the literature. Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), the rice Act1 promoter and the Figwort Mosaic Virus (FMV) 35S promoter. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants (see e.g., McElroy et al., 1990, U.S. Pat. No. 5,463,175).

In addition, it may also be preferred to bring about expression of the B. thuringiensis δ-endotoxin in specific tissues of the plant by using plant integrating vectors containing a tissue-specific promoter. Specific target tissues may include the leaf, stem, root, tuber, seed, fruit, etc., and the promoter chosen should have the desired tissue and developmental specificity. Therefore, promoter function should be optimized by selecting a promoter with the desired tissue expression capabilities and approximate promoter strength and selecting a transformant which produces the desired insecticidal activity in the target tissues. This selection approach from the pool of transformants is routinely employed in expression of heterologous structural genes in plants since there is variation between transformants containing the same heterologous gene due to the site of gene insertion within the plant genome (commonly referred to as “position effect”). In addition to promoters which are known to cause transcription (constitutive or tissue-specific) of DNA in plant cells, other promoters may be identified for use in the current invention by screening a plant cDNA library for genes which are selectively or preferably expressed in the target tissues and then determine the promoter regions.

An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The lectin protein in soybean seeds is encoded by a single gene (Le1) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990). An expression vector containing a coding region that encodes a polypeptide of interest can be engineered to be under control of the lectin promoter and that vector may be introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991). The expression of the polypeptide would then be directed specifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Other exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (McBride and Summerfelt, 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35S transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.

The promoters used in the DNA constructs of the present invention may be modified, if desired, to affect their control characteristics. For example, the CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots. The resulting chimeric promoter may be used as described herein. For purposes of this description, the phrase “CaMV35S” promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Examples of such enhancer sequences have been reported by Kay et al. (1987). Chloroplast or plastid specific promoters are known in the art (Daniell et al., U.S. Pat. No. 5,693,507; herein incorporated by reference), for example promoters obtainable from chloroplast genes, such as the psbA gene from spinach or pea, the rbcL and atpB promoter region from maize, and rRNA promoters. Any chloroplast or plastid operable promoter is within the scope of the present invention.

The RNA produced by a DNA construct of the present invention also contains a 5′ non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA. The 5′ non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5′ non-translated sequence that accompanies the promoter sequence. As shown below, a plant gene leader sequence which is useful in the present invention is the petunia heat shock protein 70 (hsp70) leader (Winter et al., 1988).

An exemplary embodiment of the invention involves the plastid targeting or plastid localization of the B. thuringiensis amino acid sequence. Plastid targeting sequences have been isolated from numerous nuclear encoded plant genes and have been shown to direct importation of cytoplasmically synthesized proteins into plastids (reviewed in Keegstra and Olsen, 1989). A variety of plastid targeting sequences, well known in the art, including but not limited to ADPGPP, EPSP synthase, or ssRUBISCO, may be utilized in practicing this invention. In alternative embodiments preferred, plastidic targeting sequences (peptide and nucleic acid) for monocotyledonous crops may consist of a genomic coding fragment containing an intron sequence as well as a duplicated proteolytic cleavage site in the encoded plastidic targeting sequences.

Tables 5-7 list promoters which are illustrative of those known in the art, but which are not meant to be limiting.

TABLE 5 PLANT PROMOTERS Promoter Reference Viral Figwort Mosaic Virus (FMV) U.S. Pat. No. 5,378,619 Cauliflower Mosaic Virus (CaMV) U.S. Pat. No. 5,530,196 U.S. Pat. No. 5,097,025 U.S. Pat. No. 5,110,732 Plant Elongation Factor U.S. Pat. No. 5,177,011 Tomato Polygalacturonase U.S. Pat. No. 5,442,052 Arabidopsis Histone H4 U.S. Pat. No. 5,491,288 Phaseolin U.S. Pat. No. 5,504,200 Group 2 U.S. Pat. No. 5,608,144 Ubiquitin U.S. Pat. No. 5,614,399 P119 U.S. Pat. No. 5,633,440 α-amylase U.S. Pat. No. 5,712,112 Viral enhancer/Plant promoter CaMV 35Senhancer/mannopine U.S. Pat. No. 5,106,739 synthase promoter

TABLE 6 TISSUE SPECIFIC PLANT PROMOTERS Tissue Specific Promoter Tissue(s) Reference Blec epidermis U.S. Pat. No. 5,646,333 malate synthase seeds; seedlings U.S. Pat. No. 5,689,040 isocitrate lyase seeds; seedlings U.S. Pat. No. 5,689,040 patatin tuber U.S. Pat. No. 5,436,393 ZRP2 root U.S. Pat. No. 5,633,363 ZRP2(2.0) root U.S. Pat. No. 5,633,363 ZRP2(1.0) root U.S. Pat. No. 5,633,363 RB7 root U.S. Pat. No. 5,459,252 root U.S. Pat. No. 5,401,836 fruit U.S. Pat. No. 4,943,674 meristem U.S. Pat. No. 5,589,583 guard cell U.S. Pat. No. 5,538,879 stamen U.S. Pat. No. 5,589,610 SodA1 pollen; middle layer, stomium Van Camp et al., 1996 of anthers SodA2 vasular bundles; stomata; Van Camp et al., 1996 axillary buds; pericycle; stomium; pollen CHS15 flowers; root tips Faktor et al., 1996 Psam-1 phloem tissue; cortex; root tips Vander et al., 1996 ACT11 elongating tissues and organs; Huang et al., 1997 pollen; ovules zmGBS pollen; endosperm Russell and Fromm, 1997 zmZ27 endosperm Russell and Fromm, 1997 osAGP endosperm Russell and Fromm, 1997 osGT1 endosperm Russell and Fromm, 1997 RolC phloem tissue; bundle sheath; Graham et al., 1997 vascular parenchyma Sh phloem tissue Graham et al., 1997 CMd endosperm Grosset et al., 1997 Bnm1 pollen Treacy et al., 1997 rice tungro bacilliform virus phloem Yin et al., 1997a; 1997b S2-RNase pollen Ficker et al., 1998 LeB4 seeds Baumlein et al., 1991 gf-2.8 seeds; seedlings Berna and Bernier, 1997

The ability to express genes in a tissue specific manner in plants has led to the production of male and female sterile plants. Generally, the production of male sterile plants involves the use of anther-specific promoters operably linked to heterologous genes that disrupt pollen formation (U.S. Pat. Nos. 5,689,051; 5,689,049; 5,659,124). U.S. Pat. No. 5,633,441 discloses a method of producing plants with female genetic sterility. The method comprises the use of style-cell, stigma-cell, or style- and stigma-cell specific promoters that express polypeptides that, when produced in the cells of the plant, kills or significantly disturbs the metabolism, functioning or development of the cells.

TABLE 7 INDUCIBLE PLANT PROMOTERS Promoter Reference heat shock promoter U.S. Pat. No. 5,447,858 Em U.S. Pat. No. 5,139,954 Adh1 Kyozoka et al., 1991 HMG2 U.S. Pat. No. 5,689,056 cinnamyl alcohol dehydrogenase U.S. Pat. No. 5,633,439 asparagine synthase U.S. Pat. No. 5,595,896 GST-II-27 U.S. Pat. No. 5,589,614

4.13.5 Chloroplast Sequestering and Targeting

Another approach for increasing expression of A+T rich genes in plants has been demonstrated in tobacco chloroplast transformation. High levels of expression of an unmodified Bacillus thuringiensis crystal protein-encoding genes in tobacco has been reported by McBride et al., (1995).

Additionally, methods of targeting proteins to the chloroplast have been developed. This technique, utilizing the pea chloroplast transit peptide, has been used to target the enzymes of the polyhydroxybutyrate synthesis pathway to the chloroplast (Nawrath et al., 1994). Also, this technique negated the necessity of modification of the coding region other than to add an appropriate targeting sequence.

U.S. Pat. No. 5,576,198 discloses compositions and methods useful for genetic engineering of plant cells to provide a method of controlling the timing or tissue pattern of expression of foreign DNA sequences inserted into the plant plastid genome. Constructs include those for nuclear transformation which provide for expression of a viral single subunit RNA polymerase in plant tissues, and targeting of the expressed polymerase protein into plant cell plastids. Also included are plastid expression constructs comprising a viral gene promoter region which is specific to the RNA polymerase expressed from the nuclear expression constructs described above and a heterologous gene of interest to be expressed in the transformed plastid cells.

4.13.6 Effects of 3′ Regions on Transgene Expression

The 3′-end regions of transgenes have been found to have a large effect on transgene expression in plants (Ingelbrecht et al., 1989). In this study, different 3′ ends were operably linked to the neomycin phosphotransferase II (NptII) reporter gene and expressed in transgenic tobacco. The different 3′ ends used were obtained from the octopine synthase gene, the 2S seed protein from Arabidopsis, the small subunit of rbcS from Arabidopsis, extension form carrot, and chalcone synthase from Antirrhinum. In stable tobacco transformants, there was about a 60-fold difference between the best-expressing construct (small subunit rbcS 3′ end) and the lowest expressing construct (shalcone synthase 3′ end).

4.14 Antibody Compositions and Methods of Making

In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to one or more of the polypeptides disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1988). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265. Antibody use is well known in the art and can be used for purification, immunoprecipitation, ELISA and western blot for resolving the presence of molecules having identifiable epitopes. Those skilled in the art would not encounter undue experimentation in using antibodies and such methods to idolate, identify, and characterize genes and proteins expressed from such genes as contemplated herein. Immuno-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.

4.15 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides of the present invention and DNA sequences which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. In particular embodiments of the invention, mutated crystal proteins are contemplated to be useful for increasing the insecticidal activity of the protein, and consequently increasing the insecticidal activity and/or expression of the recombinant transgene in a plant cell. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 8.

TABLE 8 Codon Abbre- Amino Acids viations¹ Codons Alanine Ala A GCA GCC GCG GCU Arginine Arg R AGA AGG CGA CGC CGG CGU Asparagine Asn N AAC AAU Aspartic acid Asp D GAC GAU Cysteine Cys C UGC UGU Glutamic acid Glu E GAA GAG Glutamine Gln Q CAA CAG Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Leucine Leu L UUA UUG CUA CUC CUG CUU Lysine Lys K AAA AAG Methionine Met M AUG UUG* Phenylalanine Phe F UUC UUU Proline Pro P CCA CCC CCG CCU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU Valine Val V GUA GUC GUG GUU *the codon UUG is also utilized as an initiation codon as a part of the tIC851 coding sequence ¹three letter code and corresponding single letter code abbreviations

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 Example 1 Bacillus thuringiensis Strains with Sequences Related to CryET70

We previously identified a B. thuringiensis strain expressing a protein which we designated CryET70. The CryET70 protein had effective coleopteran specific bioactivity when provided in bioassay feeding studies to western corn rootworm larvae, but not against southern corn rootworm larvae. We were interested in identifying additional B. thuringiensis strains which contained DNA encoding CryET70 and closely related genes. Colony blot hybridization experiments were completed as indicated below, using a probe prepared from cryET70 DNA. Wild-type B. thuringiensis strains were patched onto LB plates and incubated at 30° C. for four hours. A Nytran® Maximum-Strength Plus (Schleicher and Schuell, Keene, N.H.) circular (82 mm) membrane filter was then placed on the plates and the plates and filters were incubated at 25° C. overnight. The filters, which contained an exact replica of the patches, were then placed on fresh LB plates, and the filters and the original plates were incubated at 30° C. for 4 hr to allow for growth of the colonies. To release the DNA from the B. thuringiensis cells onto the nitrocellulose filter, the filters were placed, colony-side up, on Whatman 3 mM Chromatography paper (Whatman International LTD., Maidstone, England) soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. The filters were then neutralized by placing the filters, colony-side up, on Whatman paper soaked with 1 M NH₄-acetate, 0.02 M NaOH for 10 min. The filters were then rinsed in 3×SSC, 0.1% SDS, air dried, and baked for one hr at 80° C. in a vacuum oven to prepare them for hybridization.

Oligonucleotide primers were designed based on the cryET70 sequence (SEQ ID NO:1):

AM34: 5′-GACATGATTTTACTTTTAGAGC-3′ (SEQ ID NO: 3) AM43: 5′-CATCACTTTCCCCATAGC-3′ (SEQ ID NO: 4)

A PCR™ with primers AM 34 and AM 43 was used to amplify a cryET70 fragment from pEG1648 DNA. This PCR™ product was labeled with [α⁻³²P]dATP using the Prime-a-Gene® kit (Promega Corporation, Madison, Wis.) to generate a cryET70-specific probe. Hybridizations were performed overnight with the hybridization temperature at 63° C. Filters were washed in 1×SSC, 0.1% SDS at 63° C. Hybridizing colonies were detected by autoradiography using Kodak X-OMAT AR X-ray film. The results indicated that several B. thuringiensis strains in our collection contained DNA sequences which hybridized to cryET70 sequences under specified conditions. The strains identified by colony blot hybridization are listed in Table 9.

5.2 Example 2 Production of Antibody to CryET70

CryET70 specific polyclonal antibody was prepared so that proteins containing CryET70-related epitopes could be identified using immunological methods. Recombinant B. thuringiensis strain EG11839 containing plasmid pEG1648 expressing CryET70 was grown in C2 medium for four days at 25° C. The resulting spores and crystals were washed in 2.5× volume H₂O and resuspended at 1/20 the original volume in 0.005% Triton X-100®. The spore-crystal suspension was then loaded on a sucrose step gradient consisting of 79%, 72% and 55% sucrose. The gradient was spun overnight in a Beckman SW28 at 18,000 RPM. CryET70 crystals banded between the 79% and the 72% sucrose layers. CryET70 crystals were washed several times in H₂O and resuspended in 0.005% Triton X-100®. The purified crystals were then solubilized in 50 mM sodium carbonate (pH 10), 5 mM DTT, and any contaminating vegetative cells or spores were removed by centrifugation. The supernatant was neutralized with boric acid to pH8.4, and the solubilized crystals were sent to Rockland Laboratories (Gilbertsville, Pa.) for antibody production in rabbits according to standard procedures. The rabbits received two intradermal injections on days zero and seven with 50% CryET70 protein in sterile phosphate buffered saline, 50% complete Freund's adjuvant. Two additional boosts were given subcutaneously on days 14 and 28 before a test bleed on day 38. Two hundred fifty μg of CryET70 were used per rabbit for the initial injection, and 125 μg of CryET70 were used per rabbit for the subsequent boosts. On day 56 the rabbits were boosted again, as before, prior to a production bleed on day 71. The final boost was with 160 μg CryET70 on day 80, followed by a termination bleed on day 90.

5.3 Example 3 Southern and Western Blot Analyses

Strains identified in Example 1 as containing sequences related to cryET70 were examined further by Southern and Western blot analyses.

Total DNA was prepared from the strains by the following procedure. Vegetative cells were resuspended in a lysis buffer containing 50 mM glucose, 25 mM Tris-HCl (pH8.0), 10 mM EDTA, and 4 mg/ml lysozyme. The suspension was incubated at 37° C. for one hr. Following incubation, SDS was added to 1%. The suspension was then extracted with an equal volume of phenol:chloroform:isoamyl alcohol (50:48:2). DNA was precipitated from the aqueous phase by the addition of one-tenth volume 3 M sodium acetate, and two volumes of 100% ethanol. The precipitated DNA was collected with a glass rod, washed with 70% ethanol, and resuspended in dH₂O.

Total DNA was digested with EcoRI and separated on a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM Na₂EDTA, pH 8). The DNA was blotted onto an Immobilon-NC nitrocellulose filter (Millipore Corp., Bedford, Mass.) according to the method of Southern (1975). DNA was fixed to the filter by baking at 80° C. in a vacuum oven.

The blot was then hybridized with the cryET70 probe described in Example 1.

The filters were exposed to the labeled probe diluted in 3×SSC, 0.1% SDS, 10×Denhardt's reagent (0.2% bovine serum albumin (BSA), 0.2% polyvinylpyrrolidone, 0.2% Ficoll®), 0.2 mg/ml heparin and incubated overnight at 60° C. Following the incubation, the filters were washed in three changes of 3×SSC, 0.1% SDS at 60° C. The filters were blotted dry and exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.) overnight at −70° C. with an intensifying screen (Fisher Biotech, Pittsburgh, Pa.). Strains containing hybridizing DNA fragments are listed in Table 9.

For the Western blot analysis, B. thuringiensis strains were grown in C2 medium (Donovan et al., 1988) at 25° C. for four days until sporulation and cell lysis had occurred. The resulting spores and crystals were harvested by centrifugation, washed in approximately 2.5 times the original volume with H₂O, and resuspended in 0.005% Triton X-100® at one-tenth the original volume. Proteins from 10-fold concentrated cultures of the strains were run on a 10% SDS-polyacrylamide gel (Owl Separation Systems, Woburn, Mass.). Twenty μl of culture was added to 10 μl of 3× Laemmli buffer and heated at 100° C. for five minutes. Fifteen μl were loaded per lane. Following electrophoresis, the gel was blotted to nitrocellulose following standard Western blotting procedures (Towbin et al., 1979). The filter was blocked with TBSN (10 mM Tris, pH 7.8, 0.9% NaCl, 0.1% globulin-free BSA, 0.03% NaN₃)+2% BSA. The filter was then washed with TBSN twice and then incubated with anti-CryET70 rabbit antiserum diluted 1/1,000 in TBSN. The filter was then washed in TBSN and incubated with alkaline phosphatase conjugated sheep anti-rabbit IgG (1/1,000 dilution in TBSN). After washing in TBSN, proteins antigenically related to CryET70 were detected with ImmunoPure® NBT/BOP Substrate Kit (Pierce, Rockford, Ill.). B. thuringiensis strains producing proteins antigenically related to CryET70 as judged by Western blot analysis are indicated in Table 9.

5.4 Example 4 Bioassay Evaluation of B. thuringiensis Strains

Insect bioassays were used to characterize B. thuringiensis strains having activity directed against western corn rootworm larvae. B. thuringiensis strains were grown in C2 medium (Donovan et al., 1988) at 25° C. for four days at which time sporulation and lysis had occurred. The resulting spores and crystals were harvested by centrifugation, washed in approximately 2.5 times the original volume with water, and resuspended in 0.005% Triton X-100® at one-tenth the original culture volume. The spore-crystal suspensions were used directly in bioassay.

Insecticidal activity against WCRW larvae was determined via a surface contamination assay on an artificial diet (20 g agar, 50 g wheat germ, 39 g sucrose, 32 g casein, 14 g fiber, 9 g Wesson salts mix, 1 g methyl paraben, 0.5 g sorbic acid, 0.06 g cholesterol, 9 g Vaderzant's vitamin mix, 0.5 ml linseed oil, 2.5 ml phosphoric/propionic acid per liter) in a plastic feeding cup (175 mm² surface). All bioassays were performed using 128-well trays containing approximately 1 ml of diet per well with perforated mylar sheet covers (C-D International Inc., Pitman, N.J.). Thirty-two larvae (one per well) were tested per bioassay screen at 50 ul of a spore-crystal suspension per well of diet. The results of the bioassay screen are shown in Table 9.

TABLE 9 SUMMARY OF SOUTHERN, WESTERN, AND BIOASSAY ANALYSES Strains Southern blot Western blot % Control WCRW EG2929 + + 26 EG3218  +/− − 30 EG3221  +/− − 63 EG3303  +/− − 15 EG3304  +/− 0 EG3707 + − 45 EG3803 − 0 EG3953 + 100 EG3966 + − 7 EG4113 − − 40 EG4135 + + 45 EG4150 − − 64 EG4268 − + 46 EG4375 − 100 EG4447  +/− 0 EG4448 + − 100 EG4503  +/− − 56 EG4541  +/− − 72 EG4580 + + 33 EG4640 − − 95 EG4737 − − 72 EG4741 + − 73 EG5233 − − 52 EG5366 + − 69 EG5370 − − 16 EG5422 − 8

5.5 Example 5 Analysis of Wild-Type B. thuringiensis Strains

The CryET70 peptide sequence has previously been shown to share significant amino acid sequence identity with Cry22Aa. Based on the known nucleotide and amino acid sequences of CryET70 and Cry22Aa, thermal amplification primers were designed for sequences similar or identical to those of the CryET70 and Cry22Aa coding sequences.

TABLE 10 Thermal Amplification Oligonucleotide Sequence Alignment in cry22Aa and cryET70 Corresponding Position of Oligo in: Sequence (5′-3′) & cry22Aa cryET70 Oligo^(a) Corresponding SEQ ID NO (SEQ ID NO: 9) (SEQ ID NO: 1) 2270-1 GCATTTCATAGAGGATCAAT 262-281 350-369 SEQ ID N0: 5 2270-2 ATTGATCCTCTATGAAATGC 281-262 369-350 SEQ ID NO: 11 2270-3 GTTTCCCAAATGGATATCC 428-446 516-534 SEQ ID NO: 12 2270-4 GGATATCCATTTGGGAAAC 446-428 534-516 SEQ ID NO: 13 2270-5 ATCTAATAACCTACATCAGA 726-745 814-833 SEQ ID NO: 14 2270-6 TCTGATGTAGGTTATTAGAT 745-726 833-814 SEQ ID NO: 15 2270-7 TATGGGGAAAGTGATGAAAA 973-992 1061-1080 SEQ ID NO: 16 2270-8 TTTTCATCACTTTCCCCATA 992-973 1080-1061 SEQ ID NO: 6 2270-9 ATGTTGAATTAGAAATAG 1280-1297 1368-1385 SEQ ID NO: 17  2270-10 CTATTTCTAATTCAACAT 1297-1280 1385-1358 SEQ ID NO: 18  2270-11 AAGTCCTTGTTCTAGGAGAA 1481-1500 1569-1588 SEQ ID NO: 19  2270-12 TTCTCCTAGAACAAGGACTT 1500-1481 1588-1569 SEQ ID NO: 20  2270-13 TATGTATTCTATGATTCTAG 1840-1859 1928-1947 SEQ ID NO: 21  2270-14 CTAGAATCATAGAATACATA 1859-1840 1947-1928 SEQ ID NO: 22 ^(a)odd numbered oligonucleotides represent sequences identical to the indicated position for each gene (SEQ ID NO), and even numbered oligonucleotides represent sequences complementary to the indicated position for each gene (SEQ ID NO).

Even numbered oligonucleotides were paired with odd numbered oligonucleotides in various combinations in thermal amplification reactions in order to confirm the expected size of fragments from amplification of sequences from both cryET70 and cry22Aa. DNA obtained from strains EG4135 and EG4268 was also used in separate thermal reactions with all primer pairs. While all pairs produced amplification fragments from both cryET70 and cry22Aa, the only oligonucleotide primer pair which produced a product from DNA of strains EG4135 and EG4268 was the 2270-1 and 2270-8 primer pair (SEQ ID NO:5 & SEQ ID NO:6 respectively).

Amplification reactions were performed using ‘Taq-Beads’ (Pharmacia Biotech), a Stratagene Robocycler™, and the following cycling regimen: 94 C for 30 seconds, 45 C for 45 seconds, and 72 C for 1 minute for 30 cycles. Thermocycling was preceded by a 5 minute incubation at 94 C, followed by a 5 minute incubation at 72 C. The amplification products produced from strains EG4135 and EG4268 were cloned as blunt-end fragments into the SmaI site of pBluescript KSII(+) and sequenced. The sequences of the DNA inserts indicated the presence of an open reading frame (ORF) which displayed approximately 65% sequence identity to the corresponding region from either CryET70 or Cry22Aa.

5.6 Example 6 Sequence Analysis of the Full-Length Gene

Genomic DNA libraries from strains EG4135 and EG4268 were constructed in the Lambda Zap® II vector (Stratagene; La Jolla, Calif.) and used to isolate recombinant clones containing the entire ORF identified in Example 5. The ORF encodes a protein of 632 amino acids, designated tIC851. The nucleotide sequence encompassing the tIC851 gene (SEQ ID NO:7) is shown below:

AAATATTTTT AAAGGGGGAT ACGTAATTTG AATTCTAAAT CTATCATCGA AAAAGGGGTA   60 CAAGAGAATC AATATATTGA TATTCGTAAC ATATGTAGCA TTAATGGTTC TGCTAAATTT  120 GATCCTAATA CTAACATTAC AACCTTAACA GAAGCTATCA ATTCTCAAGC AGGAGCGATT  180 GCTGGAAAAA CTGCCCTAGA TATGAGACGT GATTTTACTC TCGTAGCAGA TATATACCTA  240 GGGTCTAAAA GTAGTGGAGC TGATGGTATT GCTATAGCGT TTCATAGAGG ATCAATTGGT  300 TTTATCGGTA CCATGGGTGG AGGCTTAGGG ATTCTAGGAG CACCAAACGG GATAGGATTT  360 GAAATAGATA CGTATTGGAA AGCAACTTCA GATGAAACAG GCGATTCATT TGGACATGGT  420 CAAATGAATG GAGCACATGC GGGATTTGTA AGTACAAATC GAAATGCAAG CTATTTAACA  480 GCCTTAGCTC CTATGCAAAA AATACCTGCA CCTAATAATA AATGGCGGGT TCTAACTATC  540 AATTGGGATG CGCGTAACAA CAAACTAACA GCACGGCTTC AAGAGAAAAG TAATGATGCT  600 TCTACTAGCA CTCCTAGTCC AAGATATCAA ACATGGGAAC TATTAAATCC TGCGTTTGAT  660 TTAAATCAGA AATATACTTT TATTATCGGC TCAGCTACAG GGGCTGCTAA TAACAAGCAT  720 CAGATTGGAG TTACTTTGTT TGAAGCATAC TTTACAAAAC CAACTATAGA GGCAAATCCT  780 GTTGATATTG AACTAGGCAC AGCGTTTGAT CCATTAAACC ATGAGCCAAT TGGACTCAAA  840 GCAACAGATG AAGTAGATGG AGATATAACA AAGGACATTA CGGTAGAATT TAATGACATA  900 GATACCTCCA AACCAGGTGC ATACCGTGTA ACATATAAAG TAGTAAATAG TTATGGAGAA  960 AGTGATGAGA AAACAATAGA AGTCGTAGTA TACACGAAAC CAACTATAAC TGCACATGAT 1020 ATTACGATTA AGAAAGACTT AGCATTTGAT CCATTAAACT ATGAACCAAT TGGACTCAAA 1080 GCAACCGATC CAATTGATGG AGATATAACA GATAAAATCG CTGTAAAATT TAATAATGTC 1140 GATACCTCTA AACCGGGTAA ATACCATGTA ACATATAAAG TGATAAATAG TTATGAAAAA 1200 ATTGATGAAA AAACAATAGA GGTCACAGTA TATACGAAAC CATCTATAGT GGCACATGAT 1260 GTTGAGATTA AAAAAGATAC GGCATTTGAT CCGTTAAACT ATGAACCAAT TGGGCTCAAA 1320 GCAACCGATC CAATTGATGG AGATATAACA GATAAAATTA CGGTAGAATC TAATGATGTT 1380 GATACCTCTA AACCAGGTGC ATATAGTGTG AAATATAAAG TAGTAAATAA TTATGAAGAA 1440 AGTGACGAAA AAACAATTGC CGTTACAGTA CCTGTTATAG ATGATGGGTG GGAGAATGGC 1500 GATCCGACAG GATGGAAATT CTTCTCTGGT GAAACCATTA CTCTAGAAGA TGATGAAGAG 1560 CATGCTCTTA ATGGTAAATG GGTATTTTAT GCTGATAAAC ATGTAGCAAT ATACAAACAA 1620 GTAGAGTTGA AGAATAATAT CCCTTATCAA ATTACAGTAT ATGTTAAACC AGAAGATGAA 1680 GGAACTGTGG CACACCATAT TGTTAAAGTA TCTTTCAAAT CTGATTCTGC TGGTCCAGAA 1740 AGTGAAGAAG TTATAAATGA AAGATTAATT GATGCAGAAC AGATACAAAA AGGATACAGA 1800 AAGTTAACAA GTATTCCATT TACACCAACA ACCATTGTTC CCAACAAAAA ACCAGTGATA 1860 ATTGTTGAAA ACTTTTTACC AGGATGGATA GGTGGAGTTA GAATAATTGT AGAGCCTACA 1920 AAGTAAGAAT TATAAACTAG CTTTTAATAA ATATATTTAA AAAAT 1965 The tIC851 ORF initiation codon is TTG beginning at nucleotide 28 of the sequence shown above. The deduced amino acid sequence (SEQ ID NO. 8) of the tIC851 protein is shown below, as translated from the ORF described above:

MNSKSIIEKG VQENQYIDIR NICSINGSAK FDPNTNITTL TEAINSQAGA IAGKTALDMR  60 RDFTLVADIY LGSKSSGADG IAIAFHRGSI GFIGTMGGGL GILGAPNGIG FEIDTYWKAT 120 SDETGDSFGH GQMNGAHAGF VSTNRNASYL TALAPMQKIP APNNKWRVLT INWDARNNKL 180 TARLQEKSND ASTSTPSPRY QTWELLNPAF DLNQKYTFII GSATGAANNK HQIGVTLFEA 240 YFTKPTIEAN PVDIELGTAF DPLNHEPIGL KATDEVDGDI TKDITVEFND IDTSKPGAYR 300 VTYKVVNSYG ESDEKTIEVV VYTKPTITAH DITIKKDLAF DPLNYEPIGL KATDPIDGDI 360 TDKIAVKFNN VDTSKPGKYH VTYKVINSYE KIDEKTIEVT VYTKPSIVAH DVEIKKDTAF 420 DPLNYEPIGL KATDPIDGDI TDKITVESND VDTSKPGAYS VKYKVVNNYE ESDEKTIAVT 480 VPVIDDGWEN GDPTGWKFFS GETITLEDDE EHALNGKWVF YADKHVAIYK QVELKNNIPY 540 QITVYVKPED EGTVAHHIVK VSFKSDSAGP ESEEVINERL IDAEQIQKGY RKLTSIPFTP 600 TTIVPNKKPV IIVENFLPGW IGGVRIIVEP TK 632 The predicted molecular weight for this protein is 69,398 Daltons.

The amino acid sequences of tIC851, CryET70, and Cry22Aa were aligned as shown below using the CLUSTAL alignment program (PC/GENE®). The tIC851 protein shares approximately 56% amino acid sequence identity with CryET70 and approximately 57% amino acid sequence identity with Cry22Aa. According to current Bacillus thuringiensis crystal protein nomenclature rules, the tIC851 protein should be assigned to a new secondary class of Cry proteins.

For the three way alignment, the K-tuple value was set at 1, the gap penalty value was set at 5, the window size was set at 10, the filtering level was set at 2.5, the open gap cost was set at 10, and the unit gap cost was set at 10. An “*” indicates that a position in the alignment is perfectly conserved, and a ‘.’ indicates that a position is well conserved.

Cry22Aa MKEQNLNKYDEITVQAASDYIDIRPIFQTNGSATFNSNTNITTLTQAINS  50 ET70 MKDSISKGYDEITVQA-SDYIDIRSIFQTNGSATFNSTTNITTLTQATNS  49 tIC851 MN---SKSIIEKGVQE-NQYIDIRNICSINGSAKFDPNTNITTLTEAINS  46 *.    .   * .**. ..***** *  .****.*...*******.*.** Cry22Aa QAGAIAGKTALDMRHDFTFRADIFLGTKSNGADGIAIAFHRGSIGFVGTK 100 ET70 QAGAIAGKTALDMRHDFTFRADIFLGTKSNGADGIAIAFHRGSIGFVGEK  99 tIC851 QAGAIAGKTALDMRRDFTLVADIYLGSKSSGADGIAIAFHRGSIGFIGTM  96 **************.***. ***.**.**.****************.*.. Cry22Aa GGGLGILGAPKGIGFELDTYANAPEDEVGDSFGHGAMKGSFPSFPNGYPH 150 ET70 GGGLGILGALKGIGFELDTYANAPQDEQGDSFGHGAMRGLFPGFPNGYPH 149 tIC851 GGGLGILGAPNGIGFEIDTYWKATSDETGDSFGHGQMNG---------AH 137 ********* .*****.*** .*. ** *******.*.*         .* Cry22Aa AGFVSTDKNSRWLSALAQMQRIAAPNGRWRRLEIRWDARNKELTANLQDL 200 ET70 AGFVSTDKNRGWLSALAQMQRIAAPNGRWRRLAIHWDARNKKLTANLEDL 199 tIC851 AGFVSTNRNASYLTALAPMQKIPAPNNKWRVLTINWDARNNKLTARLQE- 186 ******..*  .*.***.**.*.***..** *.*.*****..***.*.. Cry22Aa TFNDITVGEKPRTPRTATWRLVNPAFELDQKYTFVIGSATGASNNLHQIG 250 ET70 TFNDSTVLVKPRTPRYARWELSNPAFELDQKYTFVIGSATGASNNLHQIG 249 tIC851 --KSNDASTSTPSPRYQTWELLNPAFDLNQKYTFIIGSATGAANNKHQIG 234   .. ..  ....** . * * ****.*.*****.*******.** **** Cry22Aa IIEFDAYFTKPTIEANNVNVPVGATFNPKTYPGINLRATDEIDGDLTSKI 300 ET70 IIEFDAYFTKPTIEANNVSVPVGATFNPKTYPGINLRATDEIDGDLTSEI 299 tIC851 VTLFEAYFTKPTIEANPVDIELGTAFDPLNHEPIGLKATDEVDGDITKDI 284 .. *.*********** *.. .*..*.* ..  *.*.****.***.*..* Cry22Aa IVKANNVNTSKTGVYYVTYYVENSYGESDEKTIEVTVFSNPTIIASDVEI 350 ET70 IVTDNNVNTSKSGVYNVTYYVKNSYGESDEKTIEVTVFSNPTIIASDVEI 349 tIC851 TVEFNDIDTSKPGAYRVTYKVVNSYGESDEKTIEVVVYTKPTITAHDITI 334 .*. *...***.*.* *** * *************.*...***.* *..* Cry22Aa. EKGESFNPLTDSRVGLSAQDSLGNDITQNVKVKSSNVDTSKPGEYEVVFE 400 ET70 EKGESFNPLTDSRVRLSAQDSLGNDITSKVKVKSSNVDTSKPGEYDVVFE 399 tIC851 KKDLAFDPL----------------------------------NYE---- 346 .*. .*.**                                  .*. Cry22Aa VTDSFGGKAEKDFKVTVLGQPSIEANNVELEIDDSLDPLTDAKVGLRAKD 450 ET70 VTDNFGGKAEKEIKVTVLGQPSIEANDVELEIGDLFNPLTDSQVGLRAKD 449 tIC851 ------------------------------------------PIGLKATD 354                                            .**.*.* Cry22Aa SLGNDITKDIKVKFNNVDTSNSGKYEVIFEVTDRFGKKAEKSIEVLVLGE 500 ET70 SLGKDITNDVKVKSSNVDTSKPGEYEVVFEVTDRFGKKAEKSIKVLVLGE 499 tIC851 PIDGDITDKIAVKFNNVDTSKPGKYHVTYKVINSYEKIDEKTIEVTVYTK 404 ... ***... ** .*****..*.*.*...*.....* .**.*.* * .. Cry22Aa PSIEANDVEVNKGETFEPLTDSRVGLRAKDSLGNDITKDVKIKSSNVDTS 550 ET70 PSIEANNVEIEKDERFDPLTDSRVGLRAKDSLGKDITNDVKVKSSNVDTS 549 tIC851 PSIVAHDVEIKKDTAFDPLNYEPIGLKATDPIDGDITDKITVESNDVDTS 454 *** *..**..*.. *.**. ...**.*.*... ***......*..**** Cry22Aa KPGEYEVVFEVTDRFGKYVEKTIGVIVPVIDDEWEDGNVNGWKFYAGQDI 600 ET70 KPGEYEVVFEVTDRFGKYVKKLIVVIVPVIDDEWEDGNVNGWKFYAGQDI 599 tIC851 KPGAYSVKYKVVNNYEESDEKTIAVTVPVIDDGWENGDPTGWKFFSGETI 504 ***.*.* ..*......  .* * *.******.**.*. .****..*..* Cry22Aa KLLKDPDKAYKGDYVFYDSRHVAISKTIPLTDLQINTNYEITVYAKAES- 649 ET70 TLLKDPEKAYKGEYVFYDSRHAAISKTIPVTDLQVGGNYEITVYVKAES- 648 tIC851 TLEDDEEHALNGKWVFYADKHVAIYKQV---ELKNNIPYQITVYVKPEDE 551 .* .* ..* .*..***...*.** * .   .*. .  *.****.*.*. Cry22Aa ---GDHHLKVTYKKDPAGPEEPPVFNRLISTGTLVEKDYRELKGT-FRVT 695 ET70 ---GDHHLKVTYKKDPKGPEEPPVFNRLISTGKLVEKDYRELKGT-FRVT 694 tIC851 GTVAHHIVKVSFKSDSAGPESEEVINERLIDAEQIQKGYRKLTSIPFTPT 601    ..* .**..*.*. ***.  *.*  . ... ..*.**.*... *  * Cry22Aa EL--NKAPLIIVENFGAGYIGGIRIV--KIS 722 ET70 EL--NQAPLIIVENFGAGYIGGIRIV--KIS 721 tIC851 TIVPNKKPVIIVENFLPGWIGGVRIIVEPTK 632

..  *. *.****** .*.***.**.   ..?!

5.7 Example 7 Expression of the tIC851 Protein in B. thuringiensis and Bioassay Evaluation

The coding region for tIC851 was cloned into the B. thuringiensis shuttle vector pEG597 (Baum et al., 1990) together with about 0.6 kb of flanking native DNA both up and down stream of the ORF, giving rise to the recombinant plasmids pIC17501 and pIC17502. These plasmids contain a gene which confers chloramphenicol resistance on a B. thuringiensis host cell. Plasmid pMON56207, containing the cryET70 coding sequence, confers erythromycin resistance to a B. thuringiensis host. These plasmids were introduced into the Cry-B. thuringiensis strain EG10650 by electroporation. Recombinants harboring the correct plasmids were selected for growth on starch agar medium supplemented with the appropriate antibiotic.

Recombinants were grown in C2 medium for 72-96 hours, at which time the cultures were sporulated and the cells lysed. Plasmids pIC17501 and pIC17502, differing only with respect to the orientation of the tIC851 gene insert, directed the production of a protein with an apparent molecular mass of approximately 75 kDa, as judged by SDS polyacrylamide gel electrophoresis. EG10650 recombinants harboring the cloning vector pEG597 did not produce a crystal protein. Plasmid pMON56207 directed the production of CryET70, with an apparent molecular mass of approximately 80 kDa.

tIC851 was tested against boll weevil larvae and western corn rootworm (WCRW) larvae in an insect feeding bioassay and shown not to have activity against WCRW, but surprisingly good activity against boll weevil. Based on the similarity of tIC851 to CryET70 and Cry22Aa, these two proteins were also tested against boll weevil. A dose-response study on the susceptibility of the boll weevil to these B. thuringiensis toxins was performed by diet incorporation (Stone et al. 1991). A series of 3 to 8 concentrations prepared by serial dilution was used in each instance. First instar larvae were manually infested onto the diet. Mortality and weight measurements were recorded 10 days after infestation. Larvae that were dead or were still at the neonate stage were considered dead in tabulating larval responses to the individual proteins. Concentration-mortality regressions were estimated assuming the probit model (SAS software 1995). Weight records were used to calculate effective concentrations using the non-linear regression model (SAS1995).

Surprisingly, Cry22Aa was also found to have significant toxicity to boll weevil larvae comparable to that of CryET70, as indicated in Table 11. This is the first report that Cry22Aa and CryET70 have activity against this target insect pest.

TABLE 11 Cotton boll weevil Bioassay Protein LC₅₀ (μg/well) EC₅₀ (μg/well) CryET70 3.12 (1.95-5.00)  1.92 ± 0.37 Cry22Aa 0.72 (0.022-1.70) 0.36 ± 0.18

The toxin encoded by the tIC851 gene has interesting similarities as well as differences when compared with the toxins encoded by the CryET70 and Cry22Aa genes. Both CryET70 and Cry22Aa have within their primary sequence four repeating regions of approximately 80 amino acids each, aligned in a head-to-tail fashion. The sequence of tIC851 shows that the tIC851 protein has only three of the four ‘repeat domains’ found in CryET70 and Cry22Aa. This accounts for most of the approximately 90 amino acids by which the tIC851 coding sequence is shorter than that of either CryET70 or Cry22Aa. Despite this difference in structure, tIC851 has significant activity on boll weevil larvae. The novel modular structure of these three Bt toxins should be of value in semi-rational engineering of variants, which could have increased potency or spectrum of activity.

5.8 Example 8 Transgenic plants expressing tIC851

One or more transgenes, each containing a structural coding sequence of the present invention can be inserted into the genome of a plant by any suitable method such as those detailed herein. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur. Pat. Appl. Publ. No. EP0120516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen (Fromm et al., 1986; Armstrong et al., 1990; Fromm et al., 1990). For efficient expression of the polynucleotides disclosed herein in transgenic plants, the selected sequence region encoding the insecticidal polypeptide must have a suitable sequence composition (Diehn et al., 1996).

Expression of the tIC851 protein from within a plant expression vector is then confirmed in plant protoplasts by electroporation of the vector into protoplasts followed by protein blot and ELISA analysis. This vector can be introduced into the genomic DNA of plant embryos such as cotton by particle gun bombardment followed by paromomycin selection to obtain cotton plants expressing the cry gene essentially as described in U.S. Pat. No. 5,424,412. For example, the plant transformation and expression vector can be introduced via co-bombardment with a hygromycin resistance conferring plasmid into transformation susceptible cotton tissue, followed by hygromycin selection, and regeneration. Transgenic cotton lines expressing the tIC851 protein can then identified by ELISA analysis. Progeny seed from these events can then subsequently be tested for protection from susceptible insect feeding.

The B. thuringiensis polypeptides described herein are primarily localized to the cytoplasm of the plant cell, and this cytoplasmic localization results in plants that are insecticidally effective. However, in certain embodiments, it may be advantageous to direct the B. thuringiensis polypeptide to other compartments of the plant cell. Localizing B. thuringiensis proteins in compartments other than the cytoplasm may result in less exposure of the B. thuringiensis proteins to cytoplasmic proteases leading to greater accumulation of the protein yielding enhanced insecticidal activity.

Utilizing SSU CTP sequences to localize crystal proteins to the chloroplast might also be advantageous. Localization of the B. thuringiensis crystal proteins to the chloroplast could protect these from proteases found in the cytoplasm. This could stabilize the proteins and lead to higher levels of accumulation of active toxin. cry genes containing the CTP may be used in combination with the SSU promoter or with other promoters such as CaMV35S.

In addition to tIC851 expression in plants as described herein, it is specifically intended that Cry22Aa and CryET70 be used alone or in combination with each other or in combinations along with tIC851 in plants to protect plants from boll weevil infestation and in particular combinations to prevent the onset of resistance of boll weevils to any of the proteins when used alone.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented are as described in the claims below.

6.0 REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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1.-16. (canceled)
 17. A method for protecting a cotton plant from boll weevil infestation comprising providing to a boll weevil in its diet a cotton plant transformed to express a protein toxic to said weevil wherein said protein is selected from the group consisting of Cry22Aa, ET70, and tIC851.
 18. A method for protecting a cotton plant from boll weevil infestation comprising providing to a boll weevil in its diet a cotton plant or plant tissue transformed to express two or more proteins toxic to said weevil wherein said proteins are selected from the group consisting of cry22Aa, ET70, and tIC851. 19.-34. (canceled)
 35. The method according to claim 17 wherein said plant is grown in a field of cotton plants from a seed planted in said field.
 36. The method according to claim 18 wherein said plant is grown in a field of cotton plants from a seed planted in said field.
 37. The method according to claim 35 wherein said protein is tIC851.
 38. The method according to claims 36 wherein one of said proteins is tIC851. 